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Glaucoma  |   February 2015
Angiotensin II as a Morphogenic Cytokine Stimulating Fibrogenesis of Human Tenon's Capsule Fibroblasts
Author Notes
  • Huashan Hospital, Department of Ophthalmology, Fudan University, Shanghai, China 
  • Correspondence: Yiqin Xiao, Department of Ophthalmology, Huashan Hospital, Fudan University, No.12 Middle Wulumuqi Road, Shanghai 200040, China; xiaoyiqin1028@hotmail.com. Wen Ye, Department of Ophthalmology, Huashan Hospital, Fudan University, No.12 Middle Wulumuqi Road, Shanghai 200040, China; yewen0412@hotmail.com
Investigative Ophthalmology & Visual Science February 2015, Vol.56, 855-864. doi:10.1167/iovs.14-15301
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      Huimin Shi, Yuyan Zhang, Shuhao Fu, Zhaozeng Lu, Wen Ye, Yiqin Xiao; Angiotensin II as a Morphogenic Cytokine Stimulating Fibrogenesis of Human Tenon's Capsule Fibroblasts. Invest. Ophthalmol. Vis. Sci. 2015;56(2):855-864. doi: 10.1167/iovs.14-15301.

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

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Abstract

Purpose.: To examine the expression of Angiotensin II (Ang II) and its type I, and type II receptors (AT1R, AT2R) in rabbit Tenon's capsule fibroblasts after trabeculectomy, and to investigate the effects of Ang II on cultured human Tenon's capsule fibroblasts (HTFs) proliferation, migration, phenotype transition, and extracellular matrix (ECM) synthesis.

Methods.: In the rabbit, expression of Ang II, AT1R, and AT2R in Tenon's capsule fibroblasts of eyes after trabeculectomy was evaluated by immunohistochemistry. Ang II levels in aqueous humor and plasma were assessed by ELISA. Cultured HTFs, obtained from patients undergoing cataract surgery, were treated with Ang II, TGF-β1, or vehicle control. Cell proliferation and migration were evaluated by Cell Counting Kit-8 and Transwell assay, and wound scratch assay, respectively. Protein expressions of α-smooth muscle actin (α-SMA) and fibronectin (FN) were measured by Western blot and immunofluorescence. Messenger RNA expressions of α-SMA and FN were measured by real-time PCR.

Results.: In the rabbit, the expression of Ang II and AT1R increased from 1 day after surgery while AT2R increased from 7 days. In cultured HTFs, Ang II promoted cell proliferation and migration significantly (P < 0.05). Interestingly, the effect of 10−7 M Ang II was more prominent than higher concentrations (10−5 M; P < 0.05). Ang II also markedly induced the expression of α-SMA and FN, suggesting a phenotypic transition to myofibroblasts.

Conclusions.: Our results show that trabeculectomy alter the levels of Ang II and its receptors in Tenon's capsule fibroblasts, and that Ang II increase HTFs proliferation, migration, and phenotype transition, suggesting that Ang II may play a role in wound healing after trabeculectomy.

Introduction
Glaucoma is one of the leading causes of irreversible vision loss in the world.1 Elevated IOP is an important risk factor of this disease. Trabeculectomy is the standard and popular surgical method to enhance aqueous drainage and lower IOP. Despite its considerable success rate, bleb failure occurs after trabeculectomy, and finally leads to out of control IOP. Bleb failure is characterized by subconjunctival fibrosis,2 when there is a hyper cellular response, including an influx of inflammatory cells, as well as Tenon's fibroblasts proliferation, migration, phenotype transition, and extracellular matrix (ECM) deposition.3 Mitomycin C and 5-fluorouracil are used intraoperatively or postoperatively as antiscarring treatment4 to minimize fibrosis. However, complications related to the use of these compounds, such as hypotony, late-onset bleb leaks, endophthalmitis, and blebitis5 have been reported. There is a medical need to develop a safer and more efficient therapy to reduce the trabeculectomy failure rate. 
Recent evidence has suggested that the renin-angiotensin-system (RAS) is involved in the progression of fibrosis.610 The RAS is a peptidergic system with endocrine characteristics. It exists in the circulation and in tissues such as the heart, vasculature, skin, and nervous system. Angiotensinogen is produced from the liver and converted to Angiotensin (Ang) I by renin. Next, Ang I is converted to Ang II by the Ang-converting enzyme (ACE). Ang II is the main biologically active factor of RAS, which plays a critical role in regulating blood pressure. Moreover, Ang II has been reported to be associated with increased fibrosis in many tissues. An increase in Ang II level causes fibrotic disorders in the liver,11 kidney,7 cardiac muscles,12 skin,13 and many other tissues by increasing the synthesis of ECM proteins, such as fibronectin (FN), through the Ang II type 1 receptor (AT1R).14,15 Ang II activates hepatic stellate cells in the liver, which in turn induces cell proliferation,16 cell migration,17 and collagen synthesis.17 Together, these cellular effects constitute the fibrogenic actions of the Ang II peptide. In addition, Ang II causes transdifferentiation in cultured renal epithelial cells.18 Similarly, many investigators independently demonstrated the role of Ang II in cardiac fibrosis.1922 Ang II–induced fibrosis can be inhibited by AT1R antagonists, indicating that the fibrotic effects of Ang II are primarily mediated via AT1R.22,23 
Jurklies and colleagues24 showed that RAS existed in ocular structures independent of the circulating level of RAS. Expressions of angiotensin and ACE were found in human eyes, further supporting the notion that intraocular Ang II synthesis is independent the levels of renin, angiotensinogen, and ACE in the circulation.25 Furthermore, the expression of AT1R and type 2 receptors (AT2R) has been successfully detected in the eye.26,27 In addition, there exists immunohistochemical evidence of Ang II, AT1R, and AT2R in human eyes.28 In a wound healing model developed by subconjunctival blunt dissection, collagen deposition and cell infiltration were inhibited in AT1R-deficient (AT1RKO) mice, while increased in AT2R-deficient (AT2RKO) mice.29 These results indicate that Ang II and its receptors are involved in the subconjunctival fibrosis process after trabeculectomy. However, the direct effects of Ang II on filtration surgery, especially on human Tenon's fibroblasts (HTFs) are largely unknown. 
In the current study, we investigated whether the expression of Ang II and its receptors changed after trabeculectomy in the rabbit in vivo and the biological effects of Ang II on proliferation, migration, phenotype transition, and ECM synthesis of HTFs. Our overall aim was to evaluate the potential role of Ang II in wound healing after trabeculectomy. 
Materials and Methods
All animal procedures complied with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and were approved by the Institute Animal Use and Care Committee. The process of obtaining human Tenon's capsule tissue for cell culture experiments was granted hospital ethics review board approval and adhered to the Declaration of Helsinki. 
Animals
New Zealand rabbits, 3- to 5-months old and weighing 1.5 to 2.0 kg, were purchased from the Experimental Animal Center of Shanghai Medical School (Shanghai, China) and were acclimatized for 1 week before experiments. Thirty rabbits were randomly divided into a control group and a surgical group. Ear vein blood and aqueous humor samples of right eyes were extracted before the surgery from all rabbits. Standard trabeculectomy was performed on the right eyes of all the rabbits in the surgical group. At days 1, 3, 7, 14, and 28 after surgery, three rabbits of each group were randomly selected for aqueous humor sampling on the treated eye. The rabbits were euthanized and immunohistochemical examination was used for the treated eye. 
Ang II Levels of Plasma and Aqueous Humor
Plasma and aqueous humor samples were centrifuged for 15 minutes at 4000g at 4°C. The supernatants were assayed for concentration of Ang II by an Angiotensin II ELISA kit (Bioleaf, Shanghai, China) according to the manufacturer's instruction. 
Histologic Examinations
The eyes were immediately fixed after isolation with 4% formaldehyde solution for at least 24 hours. Tissue samples were then dehydrated and embedded in paraffin. Serial sections (5-μm thick) were cut, dehydrated, and stained with hematoxylin-eosin for light microscopic examination and with immunohistochemistry of Ang II, AT1R, and AT2R (1:100; Boster, Wuhan, China) at the Tenon's capsule of surgical site. Images of each group at each time point were taken from five independent fields of the bleb areas in Tenon's capsules. Numbers of first-antibody-positive fibroblasts of each image were normalized to the same numbers of total fibroblasts in each group. 
Cell Culture
Human Tenon's explants were obtained from patients during cataract surgery. These subjects, one male and two females, between 47 and 65 years of age, did not have a prior history of glaucoma or ocular surgery. Primary HTFs were generated as an expansion culture of the human Tenon's explants and were grown in Dulbecco's modified Eagle's medium (DMEM; Hyclone, Logan, UT, USA) with 10% heat-inactivated fetal bovine serum (Gibco Life Technologies, Karlsruhe, Germany), 100 U/mL penicillin and 100 mg/mL streptomycin (Biochrom, Berlin, Germany) in 5% CO2 at 37°C. Cells were maintained in the logarithmic growth phase. Cells from generations 5 to 10 were used for the experiments. Within each experiment the cells were of the same line and from the same generation. Cells were incubated to a subconfluent status (80% confluence) and starved in serum-free DMEM for 24 hours before the experiments. 
Cell Proliferation
Cell proliferation was determined by Cell Counting Kit-8 (CCK-8; Dojindo, Molecular Technologies, Inc., Gaithersburg, MD, USA). Human Tenon's fibroblasts were plated in 96-well plates at a density of 5000 cells per well (100 μL) and cultured in growth medium with TGF-β1 (10 ng/mL; PeproTech, Rocky Hill, NJ, USA) or with different concentrations of Ang II (10−9 M–10−6 M) for 24 hours. The number of cells was counted according to the protocol of the kit. 
Transwell Cell Migration Assay
Human Tenon's fibroblasts were trypsinized and resuspended at a concentration of 2.5 × 105/mL in FBS-free-DMEM. Media (500 μL) containing 10 ng/mL TGF-β1 or Ang II (10−7 M or 10−5 M) were added to a 24-well plate and an 8-μm pore size insert (BD Falcon, Franklin Lakes, NJ, USA) was added to the wells before the cells (150 μL) were placed inside the insert. After a 24-hour incubation, the under surface was gently rinsed with PBS and stained with 0.25% (wt/vol) crystal violet (Sigma-Aldrich Corp., St. Louis, MO, USA) for 15 minutes, rinsed again with sterile water, and allowed to dry. The inserts were viewed under a light microscope and the numbers of cells/field in five randomly chosen fields were counted at ×100 magnification. Images of ×200 magnification were taken for cell phenotype record. 
Scratch Wound Assay
To evaluate cell motility, an in vitro scratch wound assay was performed. There were 5 × 106 HTFs/well cultured in a 6-well plate and when cells reached 90% confluence, a single scratch was created in the center of the cell monolayers by gently scraping the attached cells with a sterile 1-mL micropipette tip. Then cells were immediately placed in serum-free media with or without TGF-β1 or Ang II. Bright-field images were obtained immediately after scraping and at various times over a span of 12 hours thereafter. The densities of cells migrating into the denuded areas were quantitated. Images of each group at each time point were taken from five independent fields of the scratched areas and ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA) was used for quantification. 
Immunofluorescence
Human Tenon's fibroblasts were seeded on coverslips in a 24-well plate with TGF-β1 (10 ng/mL) or Ang II (10−7 M) for 48 hours. Cells were fixed in cold 4% paraformaldehyde for 15 minutes, permeabilized in 0.3% Triton X-100 for 5 minutes, blocked in 2% normal goat serum (Jackson-Immuno, Hamburg, Germany) for 1 hour and conjugated with primary antibody against α-SMA (1:500; Sigma-Aldrich Corp.) overnight at 4°C. Negative controls were incubated with PBS replacing the primary antibody. After incubation with fluorescein isothiocyanate (FITC)-labeled secondary antibody for 1 hour at room temperature, coverslips were stained with 4′,6-diamidino-2-phenylindole (DAPI) for the nuclei and observed with a fluorescence microscope (Olympus, Tokyo, Japan). 
Western Blot Analysis
Total cell protein was extracted in Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing a protease inhibitor cocktail (Roche, Mannheim, Germany). The protein extracts were separated by SDS-PAGE and transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocking, the membrane was probed with primary antibody against α-SMA (1:1000), FN (1:1000, ProteinTech, Chicago, IL, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 1:2000; Millipore, Billerica, MA, USA), followed by the appropriate horseradish peroxidase (HRP)-conjugated goat anti-rabbit or goat anti-mouse secondary antibody (Millipore). Specific bands were visualized by a standard enhanced chemiluminescence procedure (Millipore). The signals were analyzed using Image-Pro Plus (version 6.0; Media Cybernetics, Inc., Rockville, MD, USA). The band densities of each sample were normalized to the GAPDH band. 
Real-Time PCR
Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. Afterward, cDNAs were synthesized using the PrimeScript RT reagent Kit (RR036; Takara, Otsu, Shiga, Japan) and then diluted 10-fold in H2O prior to their use in semiquantitative real-time PCR reactions that contained 5 μL SYBR Premix Ex Taq (Takara), 0.2 μL forward primer, 0.2 μL reverse primer, 0.2 μL ROX Reference Dye II, and 1 μL diluted cDNA. Messenger RNA expression levels were analyzed on the ABI 7500 Detection System (Applied Biosystems). The primer sets were as follows: α-SMA, 5′-ATGGTGGGAATGGGACAAAA-3′ (forward), 5′-CGTGAGCAGGGTGGGATG-3′ (reverse); FN, 5′-AATATCTCGGTGCCATTTGC-3′ (forward), 5′-AAAGGCATGAAGCACTCAA-3′ (reverse); GAPDH, 5′-CAGTGCCAGCCTCGTCTCAT-3′ (forward), 5′-AGGGGCCATCCACAGTCTTC-3′ (reverse). The parameters were set at 95°C for 30 seconds for 1 cycle, then 95°C for 5 seconds, 60°C for 34 seconds for 40 cycles. The fold change in target gene expression was analyzed using the 2−ddCt method. 
Statistical Analysis
Statistical analyses were performed using one-way ANOVA followed by the Fisher least significant difference test for comparisons among three or more groups and unpaired t-test for comparisons between two groups using SPSS 21.0 software (SPSS, Inc., Chicago, IL, USA), where P less than 0.05 was considered significant. 
Results
Expressions of Ang II, AT1R, and AT2R Increase in Tenon's Capsule After Trabeculectomy
Based on studies in the rabbit, we determined that baseline Ang II levels in plasma showed no significant difference before trabeculectomy between the control group and the surgery group (Fig. 1A), and thus could exclude systematic interference factors at the ocular level. Ang II expression in aqueous humor did not change after surgery (Fig. 1B). However, immunohistochemistry (IHC) revealed that the expression of Ang II and its receptors, AT1R and AT2R, increased in cells of the rabbit Tenon's capsule after trabeculectomy. Cells with positive immunoreactivity for Ang II and AT1R in Tenon's capsule were prominently observed on the first day after surgery and peaked on day 7, whereas AT2R expression did not increased until 7 days post surgery (Figs. 1C–H). After 7 days, the expression levels of Ang II, AT1R, and AT2R gradually decreased, but were higher than the levels before surgery. 
Figure 1
 
Increased expression of Ang II and its receptors in Tenon's capsule after trabeculectomy in the rabbit. (A) No significant difference in Ang II levels was detected in rabbit plasma between the control group and the surgery group before surgery (n = 4). (B) No significant difference of concentrations in Ang II in aqueous humor samples was detected between the control and the surgery group on 1, 3, 7, 14, and 28 days postoperatively (n = 4). (CE) Immunohistochemical staining of Ang II (C), AT1R (D), and AT2R (E) in cells in Tenon's capsule within the filtering blebs in the control or surgery groups. Positive staining appears brown. Scale bars: 500 μm. (FH) Density of cells immunostained with Ang II (F), AT1R (G), and AT2R (H). All data are presented as mean and SEM. **P < 0.01, ***P < 0.001 versus control by unpaired t-test and Fisher test.
Figure 1
 
Increased expression of Ang II and its receptors in Tenon's capsule after trabeculectomy in the rabbit. (A) No significant difference in Ang II levels was detected in rabbit plasma between the control group and the surgery group before surgery (n = 4). (B) No significant difference of concentrations in Ang II in aqueous humor samples was detected between the control and the surgery group on 1, 3, 7, 14, and 28 days postoperatively (n = 4). (CE) Immunohistochemical staining of Ang II (C), AT1R (D), and AT2R (E) in cells in Tenon's capsule within the filtering blebs in the control or surgery groups. Positive staining appears brown. Scale bars: 500 μm. (FH) Density of cells immunostained with Ang II (F), AT1R (G), and AT2R (H). All data are presented as mean and SEM. **P < 0.01, ***P < 0.001 versus control by unpaired t-test and Fisher test.
Ang II Promotes HTFs Proliferation
To investigate whether Ang II stimulates HTFs proliferation in vitro, we treated cells with Ang II (10−9 M–10−6 M) or TGF-β1 (10 ng/mL) for 24 hours. Figure 2A shows that treatment with Ang II significantly stimulated the growth of HTFs and a maximal response was obtained with 10−7 M, which increased cell number by nearly 50% more than the control group. The other three concentrations of Ang II induced approximately 10% to 20% more cell growth than the control group, an increased level similar to the TGF-β1 treated group. In a separate study, cells were incubated with 10−7 M Ang II or TGF-β1 for 12, 24, and 48 hours, respectively. As a result, cell proliferation was more promoted in the Ang II group than in the other two groups through the 48-hour incubation period and Ang II had the maximal response on HTFs at 24 hour (Fig. 2B). Thus, these results showed that Ang II increased HTFs growth in a dose- and in a time-dependent manner. Furthermore, the number of cells incubating with 10−7 M Ang II was even more than that with TGF-β1. 
Figure 2
 
Effects of Ang II on HTFs proliferation. (A) Cells treated with TGF-β1 (10 ng/mL) and different concentrations of Ang II (10−9 M–10−6 M). The OD values were measured after 24-hours treatment. The OD values of all stimulated groups significantly increased compared with the control group. The 10−7 M of Ang II group had greater cell growth promoting effect than the other three different concentrations of Ang II groups. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05 versus control by one-way ANOVA followed by Fisher test. (B) Cells incubated with TGF-β1 (10 ng/mL) or Ang II (10−7 M). The OD values were measured at 12, 24, and 48 hours after drug treatment. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05 versus control for each time point by one-way ANOVA followed by Fisher test.
Figure 2
 
Effects of Ang II on HTFs proliferation. (A) Cells treated with TGF-β1 (10 ng/mL) and different concentrations of Ang II (10−9 M–10−6 M). The OD values were measured after 24-hours treatment. The OD values of all stimulated groups significantly increased compared with the control group. The 10−7 M of Ang II group had greater cell growth promoting effect than the other three different concentrations of Ang II groups. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05 versus control by one-way ANOVA followed by Fisher test. (B) Cells incubated with TGF-β1 (10 ng/mL) or Ang II (10−7 M). The OD values were measured at 12, 24, and 48 hours after drug treatment. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05 versus control for each time point by one-way ANOVA followed by Fisher test.
Ang II Enhances HTFs Migration
Next, we assessed the effect of Ang II on HTFs migration by using (1) the transwell assay, and (2) the scratch wound assay. Thus, we included TGF-β1 (10 ng/mL) as a positive control. Our results showed that Ang II, either at 10−7 M or 10−5 M, promoted greater cell migration into the lower transwell chamber, compared with the control group of cells (Figs. 3A, B). Furthermore, low concentration of Ang II (10−7 M) induced more cells locomotion than the higher concentration did (10−5 M), and Ang II at either concentration was not as efficacious as TGF-β1. Under a higher magnification (×200), cells showed the cellular spreading features of myofibroblasts after having been incubated with TGF-β1 or Ang II, instead of spindle shaped cells (Fig. 3C). 
Figure 3
 
Effects of Ang II on HTFs migration. (AC) Transwell assay: Cells were plated in the upper chamber of filters with or without TGF-β1 (10 ng/mL) or Ang II (10−7 M or 10−5 M). Cells migrating to the underside of transwell chambers at 48 hours were imaged and quantified. (A) Representative images of the underside of transwell chambers of the different treatment groups (Scale bar: 100 μm). (B) Quantification for the cell numbers migrating to the underside of the wells. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05, ***P < 0.001 versus control by one-way ANOVA followed by Fisher test. (C) Representative transwell migration images (Scale bar: 20 μm). Stimulated cells showed the phenotype of myofibroblasts, while unstimulated control cells maintained the fibroblast phenotype. (DH) Scratch wound assay: a scratch to denude cells was made in the center of confluent HTFs. The cells were incubated with or without TGF-β1 (10 ng/mL) or Ang II (10−7 M or 10−5 M) for up to 48 hours, then imaged and quantified. (DG) Representative images of the different treatment groups at different time points after the scratch (Scale bar: 5 μm). (H) For quantification, cell numbers in the scratches of each group at each time point were counted and cell densities in the wound areas were calculated. Data from three independent experiments performed in triplicate are shown. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by two-way ANOVA.
Figure 3
 
Effects of Ang II on HTFs migration. (AC) Transwell assay: Cells were plated in the upper chamber of filters with or without TGF-β1 (10 ng/mL) or Ang II (10−7 M or 10−5 M). Cells migrating to the underside of transwell chambers at 48 hours were imaged and quantified. (A) Representative images of the underside of transwell chambers of the different treatment groups (Scale bar: 100 μm). (B) Quantification for the cell numbers migrating to the underside of the wells. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05, ***P < 0.001 versus control by one-way ANOVA followed by Fisher test. (C) Representative transwell migration images (Scale bar: 20 μm). Stimulated cells showed the phenotype of myofibroblasts, while unstimulated control cells maintained the fibroblast phenotype. (DH) Scratch wound assay: a scratch to denude cells was made in the center of confluent HTFs. The cells were incubated with or without TGF-β1 (10 ng/mL) or Ang II (10−7 M or 10−5 M) for up to 48 hours, then imaged and quantified. (DG) Representative images of the different treatment groups at different time points after the scratch (Scale bar: 5 μm). (H) For quantification, cell numbers in the scratches of each group at each time point were counted and cell densities in the wound areas were calculated. Data from three independent experiments performed in triplicate are shown. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by two-way ANOVA.
We used the scratch wound assay to assess HTFs migration in addition to the transwell assay. Scratch wounds were made in confluent monolayer of HTFs to denude the cells. Cell migration was examined in response to Ang II and TGF-β1. Migration of cells into the denuded areas was measured by quantifying images taken at 0, 12, 24, 36, and 48 hours after scratch. Increased numbers of cells that migrated in the scratched areas were obtained over time in all treatment groups. Larger numbers of cells were observed in the TGF-β1 (10 ng/mL), 10−7 M Ang II and 10−5 M Ang II groups compared with the control group by 12 hours (Figs. 3D–G). Ang II at 10−7 M was most efficacious in enhancing HTFs migration and its impact was more significant than that of TGF-β1, or 10−5 M of Ang II (Fig. 3H). 
Ang II Induces Transdifferentiation From HTFs to Myofibroblasts
Expression of α-SMA, absent in fibroblasts, is a distinctive characteristic of myofibroblasts. We detected the α-SMA protein with the use of a specific antibody. The results of our immunofluorescence studies showed that Ang II (10−7 M) and TGF-β1 (10 ng/mL) caused HTFs phenotype transition to myofibroblast after 48-hour treatment (Figs. 4A–C). The specificity of the α-SMA antibody in this study was confirmed; when it was replaced by PBS during immunofluorescence staining, the red fluorescence was undetectable (Fig. 4D). 
Figure 4
 
Expression of α-SMA as visualized by immunofluorescence in HTFs treated with or without Ang II (10−7 M) or TGF-β1 (10 ng/mL) for 48 hours. (AC) Immunofluorescence in the presence of anti–α-SMA antibody. (D) Primary anti–α-SMA antibody was replaced by PBS.
Figure 4
 
Expression of α-SMA as visualized by immunofluorescence in HTFs treated with or without Ang II (10−7 M) or TGF-β1 (10 ng/mL) for 48 hours. (AC) Immunofluorescence in the presence of anti–α-SMA antibody. (D) Primary anti–α-SMA antibody was replaced by PBS.
To further quantify the effect of Ang II on the expression of α-SMA protein and mRNA, we treated HTFs with different concentrations of Ang II (10−10 M–10−6 M) at different time periods. In Figure 5, the results show that exposure to Ang II significantly increased α-SMA protein and mRNA levels. The most prominent effect on the increased expression of α-SMA mRNA (Figs. 5A, 5B) and protein (Figs. 5C–F) was 10−7 M Ang II incubating for 36 and 48 hours. 
Figure 5
 
Ang II increased α-SMA expressions in cultured HTFs. (A) Effects of Ang II (10−10 M–10−6 M) and TGF-β1 (10 ng/mL) for 36 hours on α-SMA mRNA level. (B) Effects of 10−7 M Ang II treatment on α-SMA mRNA level for different time periods: 12, 24, 36, 48, and 72 hours. (C, E) Effects of Ang II (10−10 M–10−6 M) and TGF-β1 (10 ng/mL) for 48 hours on α-SMA protein levels. (D, F) Effects of 10−7 M Ang II treatment for different time periods on α-SMA protein level. Data represent mean and SEM from three independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by one-way ANOVA followed by Fisher's test.
Figure 5
 
Ang II increased α-SMA expressions in cultured HTFs. (A) Effects of Ang II (10−10 M–10−6 M) and TGF-β1 (10 ng/mL) for 36 hours on α-SMA mRNA level. (B) Effects of 10−7 M Ang II treatment on α-SMA mRNA level for different time periods: 12, 24, 36, 48, and 72 hours. (C, E) Effects of Ang II (10−10 M–10−6 M) and TGF-β1 (10 ng/mL) for 48 hours on α-SMA protein levels. (D, F) Effects of 10−7 M Ang II treatment for different time periods on α-SMA protein level. Data represent mean and SEM from three independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by one-way ANOVA followed by Fisher's test.
Ang II Increases Expression of an ECM Protein Fibronectin
When HTFs were treated with different concentrations of Ang II (from 10−10 M–10−6 M), FN mRNA expression increased at all doses, except for at 10−6 M (Fig. 6A). With stimulation by Ang II (10−7 M), the expression of FN mRNA increased after 12 hours and peaked at 36 hours (3-fold; Fig. 6B). At the protein level, FN expression of HTFs being incubated with Ang II increased in a time and dose-dependent manner. The FN protein expression was not significantly elevated until the concentration of Ang II ran up to 10−8 M (Fig. 6C). Under the stimulation of 10−8 M Ang II, the FN protein expression increased after 48 hours and this expression was pronounced at 72 hours (Fig. 6D). 
Figure 6
 
Increased expression of fibronectin of HTFs after Ang II incubating. (A) Effects of different doses of Ang II exposure for 36 hours on FN mRNA expression. (B) Effects of 10−7 M Ang II exposure for different time courses on FN mRNA expression. Ang II increased the expression of FN mRNA in a time dependent manner. (C, E) Dose response of Ang II–stimulated expression of FN protein. Increases in FN expression were observed in HTFs under treatment of TGF-β1 and Ang II (10−8 M and 10−7 M) for 72 hours. (D, F) Time course of α-SMA protein expression on HTFs. Data represent mean and SEM from three independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by one-way ANOVA followed by Fisher's test.
Figure 6
 
Increased expression of fibronectin of HTFs after Ang II incubating. (A) Effects of different doses of Ang II exposure for 36 hours on FN mRNA expression. (B) Effects of 10−7 M Ang II exposure for different time courses on FN mRNA expression. Ang II increased the expression of FN mRNA in a time dependent manner. (C, E) Dose response of Ang II–stimulated expression of FN protein. Increases in FN expression were observed in HTFs under treatment of TGF-β1 and Ang II (10−8 M and 10−7 M) for 72 hours. (D, F) Time course of α-SMA protein expression on HTFs. Data represent mean and SEM from three independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by one-way ANOVA followed by Fisher's test.
Discussion
As the gold-standard surgical method, trabeculectomy has been widely and confidently used in the treatment of glaucoma. However, subconjunctival fibrosis has reduced the positive outcome of patients treated with trabeculectomy, even leading to surgical failure. This study was aimed to investigate possible molecular mechanisms in subconjunctival fibrosis, which may help with the discovery of useful markers and novel therapeutic targets. In the animal model, we found that after trabeculectomy, the expression of Ang II and its receptors AT1R and AT2R in HTFs were significantly enhanced, suggesting that Ang II might be involved in subconjunctival fibrosis. Confirming the in vivo results, our in vitro results showed that Ang II promoted HTFs proliferation, migration, and transformation to myofibroblasts as well as with the upregulation of FN expression, with FN a key component of ECM. All of these cellular responses can produce subconjunctival fibrosis. 
There are four phases that are primarily involved in the wound healing process after conjunctiva injury: hemostasis, inflammatory phase, proliferative phase, and remodeling phase. Many cytokines take part in the four phases and the central mediator is TGF-β.30 Transforming growth factor–β is released from fibroblasts and inflammatory cells and is responsible for the proliferation, recruitment of fibroblasts, even the synthesis of ECM. Researchers have found the elevations of all three forms of TGF-β during the conjunctival scarring process31 and have confirmed that TGF-β induced the proliferation, differentiation of fibroblasts, and ECM deposition in vivo and in vitro.3133 Our previous studies have shown that different isoforms of TGF-β had similar actions on the cultured HTFs.30 This is the reason why we applied TGF-β1 to HTFs, as the positive control in our studies. Human Tenon's capsule fibroblasts are a type of fibroblast cell that exists in the human Tenon's capsule. These cells are the main component of scar formation, and are involved in the inflammatory phase, in cell proliferation, and in ECM creation.35 
In view of the strong scar-promoting effect of TGF-β, studies mainly focused on TGF-β inhibitors to minimize scar formation after filtration surgery.33,34,3639 However, there were no successful human clinical trials reported for its various adverse consequences. Large numbers of cytokines and proteins are involved in the downstream of TGF-β–related signal pathways. Complete restraint of TGF-β may cause serious complications, thus, a new treatment of antifibrosis is needed. In this study, we demonstrated that high expression of Ang II and its two receptors on Tenon's fibroblasts after trabeculectomy in the rabbit model, shed light on a potential new approach to reduce fibrosis. 
Ang II, the main component of the RAS, is extensively reported to participate in promoting the fibrotic process in many tissues. Our results from immunohistochemical staining showed that both AT1R and AT2R are localized to the Tenon's fibroblasts. Ang II was elevated starting at 1 day postoperative, and lasted until 28 days, suggesting that Ang II might take part across the entire fibrotic process. AT1R expression was augmented to correspond with Ang II expression, whereas AT2R expression rose only after 7 days postoperative, which indicated that Ang II exerted its function primarily through interaction with AT1R. Similarly, AT1R and AT2R were identified on interstitial fibroblasts22 and dermal fibroblasts.13 At day 7 after tissue injury, the AT2R expression was increased to a maximum of 60% AT2R mRNA-positive cells.40 Real-time PCR analysis of lung homogenates obtained from donor and Idiopathic pulmonary fibrosis (IPF) lung explants revealed an increased expression of both AT1R and AT2R in IPF. In addition, AT2R expressed at lower levels compared with AT1R.22 Furthermore, we detected Ang II expression in plasma and aqueous humor by ELISA, in order to exclude influence from the body fluid content of Ang II, and the results conformed to our expectation. However, our results showed that the level of Ang II in aqueous humor after surgery remained at a steady level. There was a dispute as to the origin of ocular RAS: from blood or from local production.4144 Angiotensinogen, renin, and ACE were detected in RPE choroid and neural retina25; this situation is a prerequisite for intraocular Ang II production in RPE choroid and retina. Furthermore, the choroid was found to exhibit the highest concentration of Ang II and the most ACE activity is in the retina; this further revealed that Ang II was produced in the posterior segment rather than in the anterior segment. The aqueous humor contained less Ang II compared with the posterior vitreous and its effect in the fibrosis process was not remarkable. Thus, it was understandable that the concentration of Ang II in the aqueous humor after surgery did not change significantly. 
Phenotype transition from fibroblasts to myofibroblasts is a key process of subconjunctival fibrosis.45 When activated, fibroblasts proliferate and migrate toward the wound site, as well as exhibit an abundant endoplasmic reticulum and Golgi associated with the synthesis and secretion of ECM including collagen type I, type III, and FN. Myofibroblasts are distinguished from fibroblasts by the presence of α-SMA, stress fibers in plasma, increased expression of ED-A fibronectin, and gap junctions.46 Tang et al.47 demonstrated that Ang II induced endothelial-to-mesenchymal transition, which was inhibited by Irbesartan, an AT1R inhibitor. Ang II was also reported to contribute to adventitial fibroblast phenotypic differentiation in an AT1R-mediated manner.8 We showed that Ang II treatment induced upregulation of α-SMA expression by immunofluorescence and apparent change of cellular morphology by crystal violet stain, indicating that a cell phenotype transition was induced by Ang II from fibroblasts to myofibroblasts. Meanwhile, after Ang II incubating for more than 36 hours, the cells expressed significantly higher levels of α-SMA protein and mRNA compared with the control groups. The results suggested that Ang II participated in subconjunctival fibrosis at least in part through promoting HTFs phenotype transition. Extracellular matrix is mainly synthesized by myofibroblasts. Thus, accompanied by myofibroblasts transformed from fibroblasts, large amount of ECM will be examined. Consistent with other results, TGF-β1 and Ang II increased FN expression in HTFs in terms of mRNA and protein levels. However, the effect of Ang II on α-SMA and FN expression was weaker than TGF-β1, which was different from its effects on proliferation and migration. Therefore, Ang II and TGF-β1 may play different roles in wound healing and fibrosis that TGF-β1 mainly serves as the indicator for phenotype transition and ECM synthesis and Ang II as the commander for cells proliferation and migration in the early stage of proliferative phase. The two factors are independent factors but may interact with each other. In myoblasts, Ang II induced the increase of TGF-β1 expression through an AT1R dependent mechanism.9 Yang et al.48 found Ang II also induced a rapid activation of Smad2/3, and showed that Ang II–induced ECM expression was positively regulated by the phosphorylated Smad2/3 but negatively regulated by Smad7. Smad2/3 and Smad7 are both critical regulators in the TGF-β–dependent Smad pathways. There also exists evidence that Ang II activates MAPK pathways.8,9,49 But no studies focus on how Ang II effects TGF-β1, and what mechanism of Ang II expression influences HTFs fibrosis. 
In conclusion, we demonstrated that Ang II is involved in the process of excrescent wound healing after trabeculectomy, the major reason of subconjunctival fibrosis, through promoting HTFs proliferation, migration and phenotype transition in vitro, and elevating Ang II and its receptors expression in vivo. Thus, Ang II might be identified as a risk factor for fibrosis of filtering blebs, and target therapy against Ang II may be a potent antifibrotic agent. Despite our promising results, further research is needed to explore the molecular signal pathways underlying the effects of Ang II on HTFs, and to choose the effective inhibitors to target Ang II, which will eventually further lead to learning how to impair fibrotic progression in surgical cases. 
Acknowledgments
Supported by National Natural Science Foundation of China Grant 81400397. 
Disclosure: H. Shi, None; Y. Zhang, None; S. Fu, None; Z. Lu, None; W. Ye, None; Y. Xiao, None 
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Footnotes
 HS and YZ contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Figure 1
 
Increased expression of Ang II and its receptors in Tenon's capsule after trabeculectomy in the rabbit. (A) No significant difference in Ang II levels was detected in rabbit plasma between the control group and the surgery group before surgery (n = 4). (B) No significant difference of concentrations in Ang II in aqueous humor samples was detected between the control and the surgery group on 1, 3, 7, 14, and 28 days postoperatively (n = 4). (CE) Immunohistochemical staining of Ang II (C), AT1R (D), and AT2R (E) in cells in Tenon's capsule within the filtering blebs in the control or surgery groups. Positive staining appears brown. Scale bars: 500 μm. (FH) Density of cells immunostained with Ang II (F), AT1R (G), and AT2R (H). All data are presented as mean and SEM. **P < 0.01, ***P < 0.001 versus control by unpaired t-test and Fisher test.
Figure 1
 
Increased expression of Ang II and its receptors in Tenon's capsule after trabeculectomy in the rabbit. (A) No significant difference in Ang II levels was detected in rabbit plasma between the control group and the surgery group before surgery (n = 4). (B) No significant difference of concentrations in Ang II in aqueous humor samples was detected between the control and the surgery group on 1, 3, 7, 14, and 28 days postoperatively (n = 4). (CE) Immunohistochemical staining of Ang II (C), AT1R (D), and AT2R (E) in cells in Tenon's capsule within the filtering blebs in the control or surgery groups. Positive staining appears brown. Scale bars: 500 μm. (FH) Density of cells immunostained with Ang II (F), AT1R (G), and AT2R (H). All data are presented as mean and SEM. **P < 0.01, ***P < 0.001 versus control by unpaired t-test and Fisher test.
Figure 2
 
Effects of Ang II on HTFs proliferation. (A) Cells treated with TGF-β1 (10 ng/mL) and different concentrations of Ang II (10−9 M–10−6 M). The OD values were measured after 24-hours treatment. The OD values of all stimulated groups significantly increased compared with the control group. The 10−7 M of Ang II group had greater cell growth promoting effect than the other three different concentrations of Ang II groups. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05 versus control by one-way ANOVA followed by Fisher test. (B) Cells incubated with TGF-β1 (10 ng/mL) or Ang II (10−7 M). The OD values were measured at 12, 24, and 48 hours after drug treatment. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05 versus control for each time point by one-way ANOVA followed by Fisher test.
Figure 2
 
Effects of Ang II on HTFs proliferation. (A) Cells treated with TGF-β1 (10 ng/mL) and different concentrations of Ang II (10−9 M–10−6 M). The OD values were measured after 24-hours treatment. The OD values of all stimulated groups significantly increased compared with the control group. The 10−7 M of Ang II group had greater cell growth promoting effect than the other three different concentrations of Ang II groups. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05 versus control by one-way ANOVA followed by Fisher test. (B) Cells incubated with TGF-β1 (10 ng/mL) or Ang II (10−7 M). The OD values were measured at 12, 24, and 48 hours after drug treatment. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05 versus control for each time point by one-way ANOVA followed by Fisher test.
Figure 3
 
Effects of Ang II on HTFs migration. (AC) Transwell assay: Cells were plated in the upper chamber of filters with or without TGF-β1 (10 ng/mL) or Ang II (10−7 M or 10−5 M). Cells migrating to the underside of transwell chambers at 48 hours were imaged and quantified. (A) Representative images of the underside of transwell chambers of the different treatment groups (Scale bar: 100 μm). (B) Quantification for the cell numbers migrating to the underside of the wells. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05, ***P < 0.001 versus control by one-way ANOVA followed by Fisher test. (C) Representative transwell migration images (Scale bar: 20 μm). Stimulated cells showed the phenotype of myofibroblasts, while unstimulated control cells maintained the fibroblast phenotype. (DH) Scratch wound assay: a scratch to denude cells was made in the center of confluent HTFs. The cells were incubated with or without TGF-β1 (10 ng/mL) or Ang II (10−7 M or 10−5 M) for up to 48 hours, then imaged and quantified. (DG) Representative images of the different treatment groups at different time points after the scratch (Scale bar: 5 μm). (H) For quantification, cell numbers in the scratches of each group at each time point were counted and cell densities in the wound areas were calculated. Data from three independent experiments performed in triplicate are shown. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by two-way ANOVA.
Figure 3
 
Effects of Ang II on HTFs migration. (AC) Transwell assay: Cells were plated in the upper chamber of filters with or without TGF-β1 (10 ng/mL) or Ang II (10−7 M or 10−5 M). Cells migrating to the underside of transwell chambers at 48 hours were imaged and quantified. (A) Representative images of the underside of transwell chambers of the different treatment groups (Scale bar: 100 μm). (B) Quantification for the cell numbers migrating to the underside of the wells. Data are mean and SEM from three independent experiments with triplicate samples. *P < 0.05, ***P < 0.001 versus control by one-way ANOVA followed by Fisher test. (C) Representative transwell migration images (Scale bar: 20 μm). Stimulated cells showed the phenotype of myofibroblasts, while unstimulated control cells maintained the fibroblast phenotype. (DH) Scratch wound assay: a scratch to denude cells was made in the center of confluent HTFs. The cells were incubated with or without TGF-β1 (10 ng/mL) or Ang II (10−7 M or 10−5 M) for up to 48 hours, then imaged and quantified. (DG) Representative images of the different treatment groups at different time points after the scratch (Scale bar: 5 μm). (H) For quantification, cell numbers in the scratches of each group at each time point were counted and cell densities in the wound areas were calculated. Data from three independent experiments performed in triplicate are shown. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by two-way ANOVA.
Figure 4
 
Expression of α-SMA as visualized by immunofluorescence in HTFs treated with or without Ang II (10−7 M) or TGF-β1 (10 ng/mL) for 48 hours. (AC) Immunofluorescence in the presence of anti–α-SMA antibody. (D) Primary anti–α-SMA antibody was replaced by PBS.
Figure 4
 
Expression of α-SMA as visualized by immunofluorescence in HTFs treated with or without Ang II (10−7 M) or TGF-β1 (10 ng/mL) for 48 hours. (AC) Immunofluorescence in the presence of anti–α-SMA antibody. (D) Primary anti–α-SMA antibody was replaced by PBS.
Figure 5
 
Ang II increased α-SMA expressions in cultured HTFs. (A) Effects of Ang II (10−10 M–10−6 M) and TGF-β1 (10 ng/mL) for 36 hours on α-SMA mRNA level. (B) Effects of 10−7 M Ang II treatment on α-SMA mRNA level for different time periods: 12, 24, 36, 48, and 72 hours. (C, E) Effects of Ang II (10−10 M–10−6 M) and TGF-β1 (10 ng/mL) for 48 hours on α-SMA protein levels. (D, F) Effects of 10−7 M Ang II treatment for different time periods on α-SMA protein level. Data represent mean and SEM from three independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by one-way ANOVA followed by Fisher's test.
Figure 5
 
Ang II increased α-SMA expressions in cultured HTFs. (A) Effects of Ang II (10−10 M–10−6 M) and TGF-β1 (10 ng/mL) for 36 hours on α-SMA mRNA level. (B) Effects of 10−7 M Ang II treatment on α-SMA mRNA level for different time periods: 12, 24, 36, 48, and 72 hours. (C, E) Effects of Ang II (10−10 M–10−6 M) and TGF-β1 (10 ng/mL) for 48 hours on α-SMA protein levels. (D, F) Effects of 10−7 M Ang II treatment for different time periods on α-SMA protein level. Data represent mean and SEM from three independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by one-way ANOVA followed by Fisher's test.
Figure 6
 
Increased expression of fibronectin of HTFs after Ang II incubating. (A) Effects of different doses of Ang II exposure for 36 hours on FN mRNA expression. (B) Effects of 10−7 M Ang II exposure for different time courses on FN mRNA expression. Ang II increased the expression of FN mRNA in a time dependent manner. (C, E) Dose response of Ang II–stimulated expression of FN protein. Increases in FN expression were observed in HTFs under treatment of TGF-β1 and Ang II (10−8 M and 10−7 M) for 72 hours. (D, F) Time course of α-SMA protein expression on HTFs. Data represent mean and SEM from three independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by one-way ANOVA followed by Fisher's test.
Figure 6
 
Increased expression of fibronectin of HTFs after Ang II incubating. (A) Effects of different doses of Ang II exposure for 36 hours on FN mRNA expression. (B) Effects of 10−7 M Ang II exposure for different time courses on FN mRNA expression. Ang II increased the expression of FN mRNA in a time dependent manner. (C, E) Dose response of Ang II–stimulated expression of FN protein. Increases in FN expression were observed in HTFs under treatment of TGF-β1 and Ang II (10−8 M and 10−7 M) for 72 hours. (D, F) Time course of α-SMA protein expression on HTFs. Data represent mean and SEM from three independent experiments performed in triplicate. *P < 0.05, **P < 0.01, ***P < 0.001 versus control by one-way ANOVA followed by Fisher's test.
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