April 2017
Volume 58, Issue 4
Open Access
Glaucoma  |   April 2017
Molecular Mechanisms Underlying the Filtration Bleb-Maintaining Effects of Suberoylanilide Hydroxamic Acid (SAHA)
Author Affiliations & Notes
  • Akiko Futakuchi
    Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Toshihiro Inoue
    Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Tomokazu Fujimoto
    Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Utako Kuroda
    Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Miyuki Inoue-Mochita
    Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Eri Takahashi
    Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Saori Ohira
    Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Hidenobu Tanihara
    Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan
  • Correspondence: Toshihiro Inoue, Department of Ophthalmology, Faculty of Life Sciences, Kumamoto University, 1-1-1 Honjo, Kumamoto 860-8556, Japan; [email protected]
Investigative Ophthalmology & Visual Science April 2017, Vol.58, 2421-2429. doi:https://doi.org/10.1167/iovs.16-21403
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      Akiko Futakuchi, Toshihiro Inoue, Tomokazu Fujimoto, Utako Kuroda, Miyuki Inoue-Mochita, Eri Takahashi, Saori Ohira, Hidenobu Tanihara; Molecular Mechanisms Underlying the Filtration Bleb-Maintaining Effects of Suberoylanilide Hydroxamic Acid (SAHA). Invest. Ophthalmol. Vis. Sci. 2017;58(4):2421-2429. https://doi.org/10.1167/iovs.16-21403.

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

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Abstract

Purpose: Suberoylanilide hydroxamic acid (SAHA) has been shown to support the maintenance of experimental filtration blebs in animal models. This study was performed to investigate the molecular mechanisms underlying the bleb-maintaining effects of SAHA in modulating wound healing activities of conjunctival fibroblasts.

Methods: Human conjunctival fibroblasts (HConFs) were pretreated with SAHA before treatment with TGF-β2. Microarray-based screening was used to investigate the gene expression profiles. Gene ontology (GO) analysis was conducted to categorize the gene functions. The expression of TGF-β–induced signaling molecules, α-smooth muscle actin, and extracellular matrix (ECM) proteins were evaluated by Western blot analyses. Multiplex immunoassay was performed to evaluate supernatant cytokine concentrations. Tube formation assay was used to evaluate angiogenesis using human umbilical vein endothelial cells.

Results: GO analysis showed that SAHA, in the presence of TGF-β2, induced changes in expression of genes involved in the TGF-β receptor signaling pathway, cell proliferation, extracellular matrix organization, inflammatory responses, and angiogenesis. Subsequent in vitro experiments showed that SAHA partly inhibited the phosphorylation of Smad2, Smad3, and Akt. SAHA pretreatment potently suppressed TGF-β2–driven cell proliferation, myofibroblast differentiation, contraction, ECM production, and angiogenic cytokine expression. The supernatant of HConFs treated with SAHA inhibited tube formation.

Conclusions: SAHA has been shown to suppress angiogenesis and activation of conjunctival fibroblasts partly via inhibition of Smad and non-Smad TGF-β signaling. This in vitro study provides new evidence for the molecular basis of the potential bleb-maintaining effects of SAHA, a novel candidate drug in modulating scar formation after glaucoma filtration surgery.

Glaucoma is the second leading cause of blindness in the world.1 For treatment of glaucoma, glaucoma filtration surgery (GFS) is regarded as a useful therapeutic modality for reduction of intraocular pressure (IOP). However, postsurgical scarring of the subconjunctival tissue directly promotes loss of filtering bleb function, resulting in IOP re-elevation after GFS.2,3 A large number of clinical studies indicate that anti-inflammatory treatment with steroids and adjunctive use of mitomycin C (or 5-fluorouracil) can maintain surgically created filtration blebs for long periods.46 However, these treatments have the potential risk of complications, such as aqueous leakage and late-onset endophthalmitis.3 Therefore, the development of a novel therapeutic modality for supporting maintenance of filtration blebs is required. 
Recent studies have highlighted the role of epigenetic modulation in organ fibrosis.7,8 Among the available epigenetic drugs, histone deacetylase (HDAC) inhibitors have been widely investigated and show antifibrotic effects in multiple organs, including the kidneys, lungs, heart, liver, skin, and cornea.914 HDAC inhibitors induce acetylation of not only histones but also nonhistone proteins, including various transcription factors, thereby altering gene expression.15 Recently, Sharma et al.16 reported that suberoylanilide hydroxamic acid (SAHA), a broad-spectrum HDAC inhibitor, successfully improved the filtering surgery outcome in a rabbit model, with prolonged bleb survival and reduction of IOP. However, the mechanisms underlying the bleb-maintaining effect of SAHA have not been elucidated. 
Wound healing at the conjunctiva is a complex process, including proliferation of fibroblasts, transition into myofibroblasts, extracellular matrix (ECM) remodeling, contraction, infiltration of inflammatory cells, and angiogenesis.3,17,18 Fibroblasts are activated by multiple chemical signals, the central mediator of which is transforming growth factor (TGF)-β19,20 and undergo differentiation into activated myofibroblasts. The aqueous humor in glaucomatous eyes is known to contain high levels of various cytokines and growth factors, especially TGF-β2.21 As trabeculectomy leads to the influx of aqueous humor into filtration blebs, it is assumed that conjunctival fibroblasts are exposed to TGF-β2, contributing to scar formation after trabeculectomy. 
Here, we report that SAHA induces dynamic changes in gene expression identified by microarray-based screening experiments and that it suppresses angiogenesis, as well as the activation of conjunctival fibroblasts partly via inhibition of Smad- and non-Smad TGF-β signaling in vitro. 
Materials and Methods
Cell Culture
Human primary conjunctival fibroblasts (HConFs; ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in fibroblast medium (ScienCell Research Laboratories) supplemented with fetal bovine serum (FBS; ScienCell Research Laboratories), fibroblast growth supplement (FGS; ScienCell Research Laboratories), and antibiotics (penicillin [100 U/mL]/streptomycin [100 μg/mL] solution; ScienCell Research Laboratories) and maintained at 37°C in a humid atmosphere of 5% CO2, as described previously.22 HConFs at passage 5 or 6 were used in all experiments. Cells were starved in serum-free Dulbecco's modified Eagle's medium (DMEM; Wako Pure Chemical Industries, Osaka, Japan) overnight before each treatment. Human umbilical vein endothelial cells (HUVECs; Lonza, Walkersville, MD, USA) were cultured in endothelial basal medium-2 (Lonza) supplemented with components of SingleQuots Kit (Lonza) and maintained under the same conditions as HConFs. HUVECs were used for tube formation assay at passages 4 to 6. 
Western Blot Analysis
For myofibroblast differentiation, HConFs were stimulated with 5 ng/mL recombinant human TGF-β2 (R&D Systems, Minneapolis, MN, USA). Prior to the addition of TGF-β2, the cells were treated with or without different concentrations of SAHA (1–5 μM; Sigma-Aldrich Corp., St. Louis, MO, USA). Protein extraction was performed at different time intervals as indicated in the figure legends. Western blot analysis was performed as described previously.22 Primary antibodies to fibronectin, type I collagen, type III collagen, and phospho-Smad3 (Ser423/425) were obtained from Abcam (Cambridge, UK), and antibodies to phospho-Smad2 (Ser465/467), Smad2, Smad3, phospho-p38 (Thr180/182), p-38, phospho-Akt (Ser473), Akt, phospho-ERK1/2 (Thr202/204), ERK1/2, and horseradish peroxidase (HRP)-linked secondary antibodies were obtained from Cell Signaling (Danvers, MA, USA). 
Immunocytochemistry
Immunocytochemistry was performed as described previously.23 Briefly, cells were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.5% Triton X-100, blocked with 10% FBS, and incubated overnight with anti–α-smooth muscle actin (α-SMA) antibody (Sigma-Aldrich Corp.) at 4°C. The cells were then probed with goat anti-mouse IgG secondary antibody–Alexa Fluor 488 conjugate (Thermo Fisher Scientific, Waltham, MA, USA). F-actin cytoskeleton was stained with tetramethylrhodamine isothiocyanate (TRITC) (Sigma-Aldrich Corp.). DAPI (4′,6-diamidino-2-phenylindole) was used for nuclear staining. The cells were observed with a fluorescence microscope (model BZ 700; Keyence, Osaka, Japan). 
Collagen Gel Contraction Assay
Collagen gels were prepared using the method described previously22,24,25 with slight modifications. Briefly, type I collagen (Nitta-Gelatin, Osaka, Japan), 10× DMEM (Sigma-Aldrich Corp.), reconstitution buffer (Nitta-Gelatin), and HConF suspension were mixed on ice, and this mixture was dispensed into each well of BSA-coated 24-well plates. After complete gelation, DMEM with or without SAHA (1–5 μM) was added to the top of each gel for 1 hour, followed by the addition of TGF-β2. Gels were released from the side of the wells with 26-gauge needles and incubated for 48 hours after initiation. 
Scratch Assay
This assay was performed as described previously26 with minor modifications. HConFs were treated with or without 5 μM SAHA for 1 hour, followed by TGF-β2 stimulation for 48 hours. The confluent monolayer cultures were scratched in a straight line across the well using a micropipette tip and a ruler. Wells were washed with DMEM, and the wound area was subsequently examined after 6, 12, and 18 hours under a light microscope. 
Cell Proliferation Assay
Cell proliferation was evaluated by a WST-8 assay following the instructions provided by Dojindo Laboratories (Kumamoto, Japan). Briefly, cells were inoculated into 96-well plates and after treatment with or without SAHA (1–5 μM) followed by TGF-β2 for 48 hours, WST-8 reagent solution (Dojindo Laboratories) was added to each well and incubated for 2 hours. The absorbance at 450 nm was measured using a microplate reader (Multiskan FC; Thermo Fisher Scientific). 
Cytotoxicity Analysis
Cytotoxicity was evaluated by Hoechst 33342 (Dojindo Laboratories) and propidium iodide (PI; Dojindo Laboratories) double staining, following the instructions provided by Dojindo Laboratories. Briefly, the cells were double-stained and incubated for 30 minutes. PI-positive cells were considered dead/damaged cells, whereas Hoechst-stained cells included live, dead, and damaged cells.27 The cells were observed under a fluorescence microscope (Keyence). 
DNA Microarray and Gene Ontology Analysis
Custom cDNA microarray analysis was performed using GeneChip technology (Affymetrix, Santa Clara, CA, USA). Total RNA was extracted from the cells using a NucleoSpin RNA kit (Takara Bio, Shiga, Japan), and biotinylated cDNA was prepared according to the standard Affymetrix protocol from 100 ng total RNA (GeneChip WT Terminal Labeling and Hybridization User Manual for use with the GeneChip WT PLUS Reagent Kit; Affymetrix). Following fragmentation, aliquots of single-stranded cDNA were hybridized on a GeneChip Human Gene 2.0 ST array. GeneChips were washed and stained in an Affymetrix Fluidics Station 450 and then scanned using a GeneChip Scanner 3000 7G. The data were analyzed with GeneSpring GX Version12.5 (RMA16). The trimmed mean was used with normalized data. Genes that showed a greater than 2-fold increase in expression in at least one of the pairwise probe comparisons were considered up-regulated, whereas those with an expression level less than 0.5-fold of the control were considered down-regulated. The affected genes were further analyzed by gene ontology (GO) analysis using LSKB software (World Fusion, Inc., Tokyo, Japan). LSKB uses Fisher's exact test to estimate multiplicity between GO functional classes of gene sets (http://www.geneontology.org, in the public domain) and up-regulated or down-regulated genes based on array results. 
Multiplex Immunoassay
Supernatants of HConF cultures were collected after 48 hours of stimulation with SAHA and/or TGF-β2 and stored at −20°C. Samples were diluted twofold, and human magnetic Luminex screening assays (R&D Systems) for IL-8, monocyte chemoattractant protein-1 (MCP-1), chemokine (C–X–C motif) ligand (CXCL)1, CXCL6, bone morphogenetic proteins (BMP)-2, platelet-derived growth factor (PDGF)-AA, and VEGF were performed according to the manufacturer's instructions. The samples were acquired using a Luminex 200TM System (Luminex), and data were analyzed with MasterPlex QT version 5.0 (Hitachi Solutions, Tokyo, Japan). 
Tube Formation Assay
Tube formation assay was performed to assess angiogenesis in vitro, following the instructions provided by Lonza. Briefly, growth factor-reduced Matrigel (BD Biosciences, San Diego, CA, USA) was loaded onto 96-well plates and incubated for 30 minutes. HUVECs were diluted in DMEM-based supernatants of SAHA- and/or TGF-β–treated HConFs and were seeded at 30,000 cells per well onto the Matrigel. Images of each well were taken after 12 hours of incubation. 
Statistical Analysis
The results are presented as means ± SEM. All experiments were repeated at least three times, and data were analyzed with the Tukey-Kramer multiple comparisons test. In all analyses, P < 0.05 was taken to indicate statistical significance. 
Results
Microarray Expression Profile and GO Analysis
Microarray analysis was performed to determine changes in gene expression profiles of HConFs after pretreatment with or without SAHA followed by stimulation with TGF-β2 for 48 hours. Among the affected genes, 619 genes showed more than 2-fold up-regulation and 454 genes were down-regulated to <0.5-fold. Some of the genes of myofibroblast-inducing factors (e.g., ACTA2, LOX, NOX4, and ITGA11)7 were up-regulated in the presence of TGF-β2, which was repressed by SAHA pretreatment (data not shown). Representative significantly up-regulated and down-regulated biological processes based on GO analysis are listed in Tables 1 and 2, respectively. SAHA, in the presence of TGF-β2 stimulation, down-regulated ‘cell proliferation’, ‘cell migration’, ‘wound healing’, and ‘angiogenesis’, whereas it up-regulated ‘inflammatory response’. ‘ECM organization’ and ‘TGF-β receptor signaling pathway’ were both up-regulated and down-regulated by SAHA. Thus, complex molecular mechanisms are elicited by SAHA. Accordingly, we conducted further experiments to elucidate these molecular mechanisms in conjunctival fibroblasts with TGF-β2 stimulation. 
Table 1
 
Representative GO Biological Processes of Genes in SAHA-Treated Cells Up-Regulated in the Presence of TGF-β2
Table 1
 
Representative GO Biological Processes of Genes in SAHA-Treated Cells Up-Regulated in the Presence of TGF-β2
Table 2
 
Representative GO Biological Processes of Genes in SAHA-Treated Cells Down-Regulated in the Presence of TGF-β2
Table 2
 
Representative GO Biological Processes of Genes in SAHA-Treated Cells Down-Regulated in the Presence of TGF-β2
SAHA Partly Inhibited Smad and Non-Smad Signaling
To investigate the possible effects of SAHA on Smad and non-Smad TGF-β signaling pathways, we performed time course analysis of phosphorylation levels by Western blotting. Phosphorylation levels of Smads (Smad2 and Smad3) and a non-Smad protein, Akt, were partly inhibited by SAHA, as shown in Figure 1. Although phosphorylation levels of p38 tended to be suppressed at 1 hour after treatment with SAHA, the effect was not so obvious at other time point. Phosphorylation of ERK1/2 was induced by TGF-β2 stimulation, which was not altered by SAHA pretreatment. Phosphorylation of JNK was not detected in the untreated control, TGF-β2, or SAHA pretreatment groups (data not shown). 
Figure 1
 
Time course analysis of phosphorylation levels of TGF-β–related signaling molecules. HConFs were pretreated with or without 5 μM SAHA for 1 hour, followed by TGF-β2 stimulation. Protein lysates were collected at different time points (0, 1, 3, 6, 12, and 24 hours), and expression levels of each protein were examined by Western blot analysis. Phosphorylation levels of Smads (Smad2 and Smad3) and a non-Smad protein, Akt, were partly inhibited by SAHA (indicated by arrows). Although phosphorylation levels of p38 tended to be suppressed at 1 hour after treatment with SAHA, the effect was not so obvious at other time points.
Figure 1
 
Time course analysis of phosphorylation levels of TGF-β–related signaling molecules. HConFs were pretreated with or without 5 μM SAHA for 1 hour, followed by TGF-β2 stimulation. Protein lysates were collected at different time points (0, 1, 3, 6, 12, and 24 hours), and expression levels of each protein were examined by Western blot analysis. Phosphorylation levels of Smads (Smad2 and Smad3) and a non-Smad protein, Akt, were partly inhibited by SAHA (indicated by arrows). Although phosphorylation levels of p38 tended to be suppressed at 1 hour after treatment with SAHA, the effect was not so obvious at other time points.
SAHA Suppressed TGF-β2–Induced Cell Proliferation Without Any Toxicity
A WST-8 assay was performed to evaluate the effects of SAHA on viable cell numbers. SAHA alone significantly suppressed the viable cell numbers in a dose-dependent manner, and further, the increase induced by TGF-β2 was also significantly reduced by SAHA pretreatment in a dose-dependent manner (Fig. 2A). To examine the cytotoxic effects of SAHA, double staining with Hoechst 33342 and PI was conducted. PI-positive (dead/damaged) cells were barely detectable following treatment with 5 μM SAHA (Fig. 2B). 
Figure 2
 
Effects of SAHA on viable cell numbers and cell toxicity. (A) HConFs were pretreated with SAHA (1–5 μM) for 1 hour and stimulated with TGF-β2 for 48 hours. Viable cell numbers were evaluated by WST-8 assay. Compared with the control, TGF-β2 significantly increased the viable cell number, which was significantly attenuated by pretreatment with SAHA in a dose-dependent manner. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE. (B) Under the same conditions, cytotoxic effects of SAHA were evaluated by double-staining with Hoechst 33342 (blue, live and dead cells) and PI (red, dead cells). PI-positive cells were barely detectable after treatment with SAHA at the doses tested in the present study. Cells were treated with 2 mM H2O2 for 4 hours as a positive control. Scale bar denotes 500 μm.
Figure 2
 
Effects of SAHA on viable cell numbers and cell toxicity. (A) HConFs were pretreated with SAHA (1–5 μM) for 1 hour and stimulated with TGF-β2 for 48 hours. Viable cell numbers were evaluated by WST-8 assay. Compared with the control, TGF-β2 significantly increased the viable cell number, which was significantly attenuated by pretreatment with SAHA in a dose-dependent manner. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE. (B) Under the same conditions, cytotoxic effects of SAHA were evaluated by double-staining with Hoechst 33342 (blue, live and dead cells) and PI (red, dead cells). PI-positive cells were barely detectable after treatment with SAHA at the doses tested in the present study. Cells were treated with 2 mM H2O2 for 4 hours as a positive control. Scale bar denotes 500 μm.
SAHA Inhibited TGF-β2–Induced Myofibroblast Differentiation and ECM Production
Pretreatment with SAHA for 1 hour before TGF-β2 stimulation significantly suppressed TGF-β2induced α-SMA expression in a dose-dependent manner on Western blot analysis, and almost complete inhibition was achieved at 5 μM (Fig. 3A). Immunocytochemistry demonstrated that SAHA inhibited TGF-β2driven α-SMA expression, which agreed with the results of Western blot analysis, and that SAHA repressed TGF-β2induced actin polymerization (Fig. 3B). Observation of cell morphology under phase-contrast light microscopy demonstrated that HConFs became broader and polygonal in shape after stimulation with TGF-β2 for 48 hours, suggesting differentiation into myofibroblasts, and with SAHA pretreatment HConFs showed an almost identical morphology to that observed in control experiments (Fig. 3B). Further, TGF-β2induced increases in levels of ECM proteins (fibronectin, type I collagen, and type III collagen) were all significantly suppressed by SAHA pretreatment (Figs. 4A–4C). Treatment with SAHA for 1 hour sufficiently induced histone H3 and H4 acetylation of HConFs, which was confirmed by Western blotting (data not shown). 
Figure 3
 
Effects of SAHA on α-SMA expression. HConFs were pretreated with SAHA (1–5 μM) for 1 hour and were stimulated with 5 ng/mL TGF-β2 for 48 hours. The samples were evaluated by Western blotting (A) and immunocytochemistry (B). (A) Compared with the control, TGF-β2 increased α-SMA expression, and pretreatment with SAHA inhibited the TGF-β2–induced α-SMA expression in a dose-dependent manner. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE. (B) TGF-β2–induced α-SMA expression (green) was reduced by SAHA pretreatment, and TGF-β2–induced morphologic changes were inhibited by SAHA, with the cells showing almost the same appearance as unstimulated controls. Scale bar denotes 500 μm.
Figure 3
 
Effects of SAHA on α-SMA expression. HConFs were pretreated with SAHA (1–5 μM) for 1 hour and were stimulated with 5 ng/mL TGF-β2 for 48 hours. The samples were evaluated by Western blotting (A) and immunocytochemistry (B). (A) Compared with the control, TGF-β2 increased α-SMA expression, and pretreatment with SAHA inhibited the TGF-β2–induced α-SMA expression in a dose-dependent manner. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE. (B) TGF-β2–induced α-SMA expression (green) was reduced by SAHA pretreatment, and TGF-β2–induced morphologic changes were inhibited by SAHA, with the cells showing almost the same appearance as unstimulated controls. Scale bar denotes 500 μm.
Figure 4
 
Effects of SAHA on ECM expression. HConFs were pretreated with 5 μM SAHA for 1 hour and stimulated with TGF-β2 for 48 hours. (A) Fibronectin, (B) type I collagen, and (C) type III collagen expression were evaluated by Western blotting. Compared with the control, TGF-β2 increased expression of each ECM component, and pretreatment with SAHA attenuated the TGF-β2-induced increased expression levels. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE.
Figure 4
 
Effects of SAHA on ECM expression. HConFs were pretreated with 5 μM SAHA for 1 hour and stimulated with TGF-β2 for 48 hours. (A) Fibronectin, (B) type I collagen, and (C) type III collagen expression were evaluated by Western blotting. Compared with the control, TGF-β2 increased expression of each ECM component, and pretreatment with SAHA attenuated the TGF-β2-induced increased expression levels. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE.
SAHA Suppressed TGF-β2–Induced Collagen Gel Contraction Mediated by HConFs
Collagen gel contraction assay was performed to investigate the potential effects of SAHA on contractile function. Stimulation with TGF-β2 resulted in significant gel contraction, which was potently suppressed by SAHA pretreatment in a dose-dependent manner (Figs. 5A, 5B). 
Figure 5
 
Effects of SAHA on HConF-mediated collagen gel contraction and scratch assay. (A) HConFs were pretreated with SAHA (1–5 μM) for 1 hour and stimulated with TGF-β2 for up to 48 hours. Dotted lines indicate the edges of the collagen gels. (B) The extent of gel contraction was assessed by the gel surface area. Treatment with TGF-β2 alone caused significant collagen gel contraction compared with the control, 3 μM SAHA, and 5 μM SAHA. *P < 0.01. Data are shown as means ± SE. (C) The effects of SAHA on wound healing in vitro were evaluated by scratch assay. Confluent cultures of HConFs were scratched in a straight line across the wells, and medium was replaced with fresh DMEM. HConFs were pretreated with 5 μM SAHA for 1 hour and stimulated with TGF-β2 for 48 hours. Representative phase-contrast images of the scratched explants were acquired. (D) The shortest distances between the edges of migrated cells (including their protrusions) from both sides were measured. Compared with the control, TGF-β2 stimulation caused significant cell migration 12 and 18 hours after scratching, which was attenuated by SAHA pretreatment. Scale bar denotes 500 μm. *P < 0.05 compared with control and SAHA + TGF-β2. Data are shown as means ± SE.
Figure 5
 
Effects of SAHA on HConF-mediated collagen gel contraction and scratch assay. (A) HConFs were pretreated with SAHA (1–5 μM) for 1 hour and stimulated with TGF-β2 for up to 48 hours. Dotted lines indicate the edges of the collagen gels. (B) The extent of gel contraction was assessed by the gel surface area. Treatment with TGF-β2 alone caused significant collagen gel contraction compared with the control, 3 μM SAHA, and 5 μM SAHA. *P < 0.01. Data are shown as means ± SE. (C) The effects of SAHA on wound healing in vitro were evaluated by scratch assay. Confluent cultures of HConFs were scratched in a straight line across the wells, and medium was replaced with fresh DMEM. HConFs were pretreated with 5 μM SAHA for 1 hour and stimulated with TGF-β2 for 48 hours. Representative phase-contrast images of the scratched explants were acquired. (D) The shortest distances between the edges of migrated cells (including their protrusions) from both sides were measured. Compared with the control, TGF-β2 stimulation caused significant cell migration 12 and 18 hours after scratching, which was attenuated by SAHA pretreatment. Scale bar denotes 500 μm. *P < 0.05 compared with control and SAHA + TGF-β2. Data are shown as means ± SE.
SAHA Suppressed TGF-β2–Induced Wound Healing In Vitro
Scratch assay was performed to examine the effects of SAHA on cell migration, proliferation, and wound closure. At 12 and 18 hours after scratching, the migration of HConFs across the wound chasm was accelerated in experiments with TGF-β2 treatment compared with controls, and this effect was significantly suppressed by pretreatment with 5 μM SAHA (Figs. 5C, 5D). 
Effects of SAHA on the Expression of Angiogenic Factors and Inflammatory Cytokines
For some of the affected gene products identified by GO analysis, multiplex immunoassay was performed to evaluate supernatant cytokine concentrations in HConFs (Fig. 6). PDGF-AA and VEGF, which are representative angiogenic factors, were significantly induced by TGF-β2 stimulation and were markedly inhibited by SAHA pretreatment. On the other hand, IL-8 and MCP-1, which were detected as up-regulated inflammatory cytokines in GO analysis, were indeed significantly increased by SAHA both in the presence and absence of TGF-β2, whereas CXCL1, CXCL6, and BMP2, which were also identified as up-regulated inflammatory cytokines in GO analysis, were unaffected by SAHA. Although treatment with TGF-β2 alone did not significantly change the IL-8 or MCP-1 levels, TGF-β2 differentially affected IL-8 and MCP-1 levels in the presence of SAHA; TGF-β2 significantly increased IL-8 levels in the presence of SAHA, whereas TGF-β2 significantly decreased MCP-1 levels. 
Figure 6
 
Effects of SAHA on cytokine concentrations. HConFs treated with or without 5 μM SAHA for 1 hour followed by TGF-β2 for 48 hours, and supernatants were collected to evaluate the cytokine concentrations affected in GO analysis using multiplex immunoassay. Among the inflammatory cytokines, IL-8 and MCP-1 were indeed significantly increased by SAHA, both in the presence and absence of TGF-β2, whereas CXCL1, CXCL6, and BMP2 were unaffected by SAHA. Although treatment with TGF-β2 alone did not significantly change IL-8 or MCP-1 levels, TGF-β2 differently affected IL-8 and MCP-1 levels in the presence of SAHA; TGF-β2 significantly increased IL-8 levels in the presence of SAHA, whereas TGF-β2 significantly decreased MCP-1 levels. On the other hand, TGF-β2 induced the angiogenic factors, PDGF-AA and VEGF, which were markedly repressed by SAHA. *P < 0.05, **P < 0.001, ***P < 0.0001. Data are shown as means ± SE.
Figure 6
 
Effects of SAHA on cytokine concentrations. HConFs treated with or without 5 μM SAHA for 1 hour followed by TGF-β2 for 48 hours, and supernatants were collected to evaluate the cytokine concentrations affected in GO analysis using multiplex immunoassay. Among the inflammatory cytokines, IL-8 and MCP-1 were indeed significantly increased by SAHA, both in the presence and absence of TGF-β2, whereas CXCL1, CXCL6, and BMP2 were unaffected by SAHA. Although treatment with TGF-β2 alone did not significantly change IL-8 or MCP-1 levels, TGF-β2 differently affected IL-8 and MCP-1 levels in the presence of SAHA; TGF-β2 significantly increased IL-8 levels in the presence of SAHA, whereas TGF-β2 significantly decreased MCP-1 levels. On the other hand, TGF-β2 induced the angiogenic factors, PDGF-AA and VEGF, which were markedly repressed by SAHA. *P < 0.05, **P < 0.001, ***P < 0.0001. Data are shown as means ± SE.
Supernatant of HConFs Treated by SAHA Inhibited Tube Formation of HUVECs
To investigate the antiangiogenic potential of SAHA, in vitro tube formation assay was conducted. The supernatant of TGF-β2treated HConFs induced tube formation of HUVECs, which was inhibited markedly by SAHA pretreatment (Fig. 7). 
Figure 7
 
Effects of SAHA on angiogenesis. HConFs were pretreated with or without 5 μM SAHA, followed by TGF-β2 for 48 hours. Supernatants were collected, and HUVECs were seeded onto Matrigel-coated 96-well plates with each supernatant. Phase-contrast images of tube formation of HUVECs were taken after 12 hours. Supernatant of TGF-β2–treated HConFs induced tube formation of HUVECs, which was potently inhibited by SAHA pretreatment.
Figure 7
 
Effects of SAHA on angiogenesis. HConFs were pretreated with or without 5 μM SAHA, followed by TGF-β2 for 48 hours. Supernatants were collected, and HUVECs were seeded onto Matrigel-coated 96-well plates with each supernatant. Phase-contrast images of tube formation of HUVECs were taken after 12 hours. Supernatant of TGF-β2–treated HConFs induced tube formation of HUVECs, which was potently inhibited by SAHA pretreatment.
Discussion
Despite improvements in surgical equipment and techniques, fibrosis at the surgical site remains the major cause of GFS failure; there have been a number of studies regarding therapeutic approaches to modulate the wound healing process. Sharma et al.16 reported that subconjunctival injection of SAHA 30 minutes before GFS effectively prevented postoperative fibrosis in a rabbit model and that SAHA treatment improved bleb survival and reduced bleb vascularity, IOP, α-SMA, collagen deposition, and F-actin at the surgical site. These findings suggest that epigenetic modulation (HDAC inhibition) enhances bleb survival. Therefore, the present study was performed to investigate the molecular mechanisms related to wound healing processes associated with SAHA. To our knowledge, this is the first report regarding the underlying molecular mechanisms of the antifibrotic and antiangiogenic effects of SAHA on TGF-β2stimulated human conjunctival fibroblasts in the peer-reviewed journals. 
Microarray and GO analyses were performed to exhaustively screen for genes expression of which was affected by SAHA in the presence of TGF-β2 stimulation. The results identified 619 genes with greater than 2-fold increases in expression and 454 genes that showed a reduction in expression to less than 0.5-fold, suggesting that treatment with SAHA results in complex gene interactions of TGF-β2–associated wound healing in conjunctival fibroblasts. Gene ontology analysis indicated that ‘cell proliferation’, ‘wound healing’, ‘cell migration’, and ‘positive regulation of MAP kinase activity’ were down-regulated by SAHA, consistent with the experimental results discussed below. ‘ECM organization’ and ‘TGF-β receptor signaling pathway’ were both up-regulated and down-regulated by SAHA. Notably, the expression of genes associated with ‘angiogenesis’ (PDGF-AA and VEGFA) was down-regulated by SAHA and, surprisingly, the expression of genes associated with ‘inflammatory response’ (e.g., IL-8, MCP-1, CXCL1, CXCL6, and BMP2) was up-regulated by SAHA. These data clearly showed that SAHA induced extensive molecular changes. These findings were further confirmed by in vitro experiments as follows. 
Signaling pathways driven by TGF-β are generally divided into Smad-dependent (canonical) and Smad-independent (noncanonical) pathways, including mitogen-activating protein (MAP) kinase pathways and phosphatidylinositol-3-kinase (PI3K)/AKT pathways.28 TGF-β2 induced phosphorylation of Smads (Smad2 and Smad3), as well as non-Smad proteins (p38, ERK, and Akt). Phosphorylation of Smad2, Smad3, and Akt was partly inhibited by SAHA pretreatment, although the time windows of the inhibitory effect varied between molecules. Previous studies showed that SAHA attenuates TGF-β–induced phosphorylation of Smad2 and Smad3 but not of p38 in canine corneal fibroblasts.29 In addition, Barter et al.30 reported that HDAC inhibitors reduce TGF-β–induced ERK, Akt, and JNK phosphorylation but do not affect Smad signaling in murine embryonic fibroblasts. This discrepancy may have been due to differences in animal species and/or cell origin used for the experiments. Among these signaling molecules, SAHA particularly inhibited the phosphorylation of Akt in the present study. The PI3K/Akt pathway is important for TGF-β–induced differentiation from mesenchymal cells into smooth muscle cells.31 HDAC inhibitors, including SAHA, were confirmed to reduce Akt phosphorylation in various cell types.32,33 The dephosphorylation of Akt by HDAC inhibitors is caused by blocking interactions of protein phosphatase 1 (PP1)–HDAC complex, resulting in PP1–Akt association and Akt dephosphorylation.33,34 
Compared with unstimulated fibroblasts, activated myofibroblasts are characterized by enhanced cell contractility, cell motility, cell proliferation, and production of ECM components, representative of which are fibronectin, type I collagen, and type III collagen.18 Aberrant ECM production and its subsequent contraction are important events for the formation of rigid cicatricial tissue.35 In the present study, WST-8 assay indicated that TGF-β2 increased the number of viable HConF cells, which was reduced by SAHA. PI-positive (dead/damaged) cells were barely detectable in SAHA-treated fibroblasts, excluding the possibility of cytotoxic effects. In addition, our results showed that TGF-β2 stimulation up-regulated expression of α-SMA in HConFs, indicating the fibroblast-to-myofibroblast differentiation and that the increase was suppressed by SAHA in a dose-dependent manner, suggesting antifibrotic effects of SAHA in vitro. Indeed, in the present study, up-regulated expression of the primary ECM proteins produced by myofibroblasts was observed after TGF-β2 stimulation; this effect was attenuated by SAHA. Similarly, SAHA significantly attenuated TGF-β2–induced collagen gel contraction. Scratch assay was performed to examine the effects of SAHA on cell migration and proliferation, and SAHA significantly inhibited TGF-β2-induced wound closure. Taken together, SAHA was indicated to suppress the TGF-β2–induced differentiation of HConFs to myofibroblasts and the changes in cell properties, highlighting potential usefulness for adjunctive therapy in GFS for the maintenance of filtration blebs. 
To verify the affected gene expression caused by SAHA regarding inflammation and angiogenesis, we measured cytokine levels in the HConF supernatant. The results of multiplex immunoassay indicated that SAHA actually increased IL-8 and MCP-1 protein levels, suggesting potential augmentation of the inflammatory response. In contrast, other inflammatory cytokines, CXCL1, CXCL6, and BMP2, were unaffected by SAHA. Previous studies demonstrated that two HDAC inhibitors, SAHA and trichostatin A (TSA), up-regulated IL-8 gene expression in NIH 3T3 cells by activating NF-κB signaling.36,37 In contrast, there were other contradictory reports that HDAC inhibitors (including SAHA) have anti-inflammatory effects. For example, Wang et al.38 reported that SAHA inhibited cytokine secretion (TNF-α, IL-8, IL-13, and IL-10) from human peripheral blood mononuclear cells and lymphocytes. Similarly, Choo et al.39 reported that SAHA suppressed IL-1, IL-6, IL-18, and TNF-α secretion in LPS-stimulated THP-1 monocytic cells. Interestingly, TGF-β2 differentially affected IL-8 and MCP-1 levels in the presence of SAHA, although treatment with TGF-β2 alone did not significantly alter the IL-8 or MCP-1 levels; TGF-β2 significantly increased IL-8 levels in the presence of SAHA, whereas TGF-β2 significantly decreased MCP-1 levels. This discrepancy may be due to differences in their transcriptional regulation. IL-8 is regulated by the transcription factors NF-κB, activating protein (AP-1), CAAT/enhancer-binding protein β (C/EBPβ, also known as NF-IL-6), C/EBP homologous protein (CHOP), and cAMP response element binding protein (CREB),40 whereas MCP-1 is regulated by NF-κB, AP-1, and Sp1.41 As HDAC regulate the expression of not only histones, but also nonhistone proteins including various transcription factors, it is reasonable to suggest that treatment with TGF-β2 may result in differential expression of cytokines in the presence of SAHA. Thus, it is suggested that SAHA (and other HDAC inhibitors) have complex effects on inflammatory responses, probably depending on the experimental conditions and cell types. Although our results clearly showed that the levels of IL-8 and MCP-1 secretion by cultured HConFs were increased by SAHA treatments, previous animal experiments for GFS showed no inflammation by subconjunctival injection of SAHA.16 This discrepancy may be explained by the fact that, in wound healing of GFS, inflammatory cytokines are produced mainly by macrophages, lymphocytes, and platelets, or that postoperative steroid treatments diminished the effect in the animal model. That is, minor production of inflammatory cytokines by fibroblasts may be masked by major secretion from infiltrated inflammatory cells and/or postoperative anti-inflammatory medication in animal experiments. However, as previous reports suggested complicated effects of SAHA on inflammatory responses, further studies are required to fully understand the role of SAHA in inflammatory response after GFS. 
Our results showed that SAHA markedly suppressed the TGF-β2–induced angiogenic cytokines, PDGF-AA and VEGF, in the HConF supernatant. These findings were consistent with those of a previous in vivo study of GFS, in which bleb vascularity was decreased.16 In nonocular tissues, it was reported that SAHA repressed production of angiogenic factors (e.g., VEGF, PDGF, HIF-1α, bFGF, EGF) in vitro as well as in vivo.4244 Furthermore, our tube formation assay demonstrated that the supernatant of HConFs treated by SAHA inhibited angiogenic activity of those treated with TGF-β2. As anti-VEGF treatments were shown to improve the surgical success rate of GFS in animal experiments and clinical studies by reducing bleb vascularity,45,46 our findings suggest that SAHA may support the maintenance of filtration blebs by its antiangiogenic effects in addition to its antifibrotic effects. 
In conclusion, SAHA has been shown to suppress angiogenesis and the activation of conjunctival fibroblasts partly via inhibition of Smad and non-Smad TGF-β signaling, highlighting its therapeutic potential in modulating scar formation after GFS. 
Acknowledgments
Supported by JSPS KAKENHI Grant Numbers 26293375, 15K15636, and 26462664. The funding organization had no role in the design or conduct of this research. 
Disclosure: A. Futakuchi, None; T. Inoue, None; T. Fujimoto, None; U. Kuroda, None; M. Inoue-Mochita, None; E. Takahashi, None; S. Ohira, None; H. Tanihara, None 
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Figure 1
 
Time course analysis of phosphorylation levels of TGF-β–related signaling molecules. HConFs were pretreated with or without 5 μM SAHA for 1 hour, followed by TGF-β2 stimulation. Protein lysates were collected at different time points (0, 1, 3, 6, 12, and 24 hours), and expression levels of each protein were examined by Western blot analysis. Phosphorylation levels of Smads (Smad2 and Smad3) and a non-Smad protein, Akt, were partly inhibited by SAHA (indicated by arrows). Although phosphorylation levels of p38 tended to be suppressed at 1 hour after treatment with SAHA, the effect was not so obvious at other time points.
Figure 1
 
Time course analysis of phosphorylation levels of TGF-β–related signaling molecules. HConFs were pretreated with or without 5 μM SAHA for 1 hour, followed by TGF-β2 stimulation. Protein lysates were collected at different time points (0, 1, 3, 6, 12, and 24 hours), and expression levels of each protein were examined by Western blot analysis. Phosphorylation levels of Smads (Smad2 and Smad3) and a non-Smad protein, Akt, were partly inhibited by SAHA (indicated by arrows). Although phosphorylation levels of p38 tended to be suppressed at 1 hour after treatment with SAHA, the effect was not so obvious at other time points.
Figure 2
 
Effects of SAHA on viable cell numbers and cell toxicity. (A) HConFs were pretreated with SAHA (1–5 μM) for 1 hour and stimulated with TGF-β2 for 48 hours. Viable cell numbers were evaluated by WST-8 assay. Compared with the control, TGF-β2 significantly increased the viable cell number, which was significantly attenuated by pretreatment with SAHA in a dose-dependent manner. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE. (B) Under the same conditions, cytotoxic effects of SAHA were evaluated by double-staining with Hoechst 33342 (blue, live and dead cells) and PI (red, dead cells). PI-positive cells were barely detectable after treatment with SAHA at the doses tested in the present study. Cells were treated with 2 mM H2O2 for 4 hours as a positive control. Scale bar denotes 500 μm.
Figure 2
 
Effects of SAHA on viable cell numbers and cell toxicity. (A) HConFs were pretreated with SAHA (1–5 μM) for 1 hour and stimulated with TGF-β2 for 48 hours. Viable cell numbers were evaluated by WST-8 assay. Compared with the control, TGF-β2 significantly increased the viable cell number, which was significantly attenuated by pretreatment with SAHA in a dose-dependent manner. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE. (B) Under the same conditions, cytotoxic effects of SAHA were evaluated by double-staining with Hoechst 33342 (blue, live and dead cells) and PI (red, dead cells). PI-positive cells were barely detectable after treatment with SAHA at the doses tested in the present study. Cells were treated with 2 mM H2O2 for 4 hours as a positive control. Scale bar denotes 500 μm.
Figure 3
 
Effects of SAHA on α-SMA expression. HConFs were pretreated with SAHA (1–5 μM) for 1 hour and were stimulated with 5 ng/mL TGF-β2 for 48 hours. The samples were evaluated by Western blotting (A) and immunocytochemistry (B). (A) Compared with the control, TGF-β2 increased α-SMA expression, and pretreatment with SAHA inhibited the TGF-β2–induced α-SMA expression in a dose-dependent manner. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE. (B) TGF-β2–induced α-SMA expression (green) was reduced by SAHA pretreatment, and TGF-β2–induced morphologic changes were inhibited by SAHA, with the cells showing almost the same appearance as unstimulated controls. Scale bar denotes 500 μm.
Figure 3
 
Effects of SAHA on α-SMA expression. HConFs were pretreated with SAHA (1–5 μM) for 1 hour and were stimulated with 5 ng/mL TGF-β2 for 48 hours. The samples were evaluated by Western blotting (A) and immunocytochemistry (B). (A) Compared with the control, TGF-β2 increased α-SMA expression, and pretreatment with SAHA inhibited the TGF-β2–induced α-SMA expression in a dose-dependent manner. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE. (B) TGF-β2–induced α-SMA expression (green) was reduced by SAHA pretreatment, and TGF-β2–induced morphologic changes were inhibited by SAHA, with the cells showing almost the same appearance as unstimulated controls. Scale bar denotes 500 μm.
Figure 4
 
Effects of SAHA on ECM expression. HConFs were pretreated with 5 μM SAHA for 1 hour and stimulated with TGF-β2 for 48 hours. (A) Fibronectin, (B) type I collagen, and (C) type III collagen expression were evaluated by Western blotting. Compared with the control, TGF-β2 increased expression of each ECM component, and pretreatment with SAHA attenuated the TGF-β2-induced increased expression levels. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE.
Figure 4
 
Effects of SAHA on ECM expression. HConFs were pretreated with 5 μM SAHA for 1 hour and stimulated with TGF-β2 for 48 hours. (A) Fibronectin, (B) type I collagen, and (C) type III collagen expression were evaluated by Western blotting. Compared with the control, TGF-β2 increased expression of each ECM component, and pretreatment with SAHA attenuated the TGF-β2-induced increased expression levels. *P < 0.01 compared with control, #P < 0.01 compared with TGF-β2. Data are shown as means ± SE.
Figure 5
 
Effects of SAHA on HConF-mediated collagen gel contraction and scratch assay. (A) HConFs were pretreated with SAHA (1–5 μM) for 1 hour and stimulated with TGF-β2 for up to 48 hours. Dotted lines indicate the edges of the collagen gels. (B) The extent of gel contraction was assessed by the gel surface area. Treatment with TGF-β2 alone caused significant collagen gel contraction compared with the control, 3 μM SAHA, and 5 μM SAHA. *P < 0.01. Data are shown as means ± SE. (C) The effects of SAHA on wound healing in vitro were evaluated by scratch assay. Confluent cultures of HConFs were scratched in a straight line across the wells, and medium was replaced with fresh DMEM. HConFs were pretreated with 5 μM SAHA for 1 hour and stimulated with TGF-β2 for 48 hours. Representative phase-contrast images of the scratched explants were acquired. (D) The shortest distances between the edges of migrated cells (including their protrusions) from both sides were measured. Compared with the control, TGF-β2 stimulation caused significant cell migration 12 and 18 hours after scratching, which was attenuated by SAHA pretreatment. Scale bar denotes 500 μm. *P < 0.05 compared with control and SAHA + TGF-β2. Data are shown as means ± SE.
Figure 5
 
Effects of SAHA on HConF-mediated collagen gel contraction and scratch assay. (A) HConFs were pretreated with SAHA (1–5 μM) for 1 hour and stimulated with TGF-β2 for up to 48 hours. Dotted lines indicate the edges of the collagen gels. (B) The extent of gel contraction was assessed by the gel surface area. Treatment with TGF-β2 alone caused significant collagen gel contraction compared with the control, 3 μM SAHA, and 5 μM SAHA. *P < 0.01. Data are shown as means ± SE. (C) The effects of SAHA on wound healing in vitro were evaluated by scratch assay. Confluent cultures of HConFs were scratched in a straight line across the wells, and medium was replaced with fresh DMEM. HConFs were pretreated with 5 μM SAHA for 1 hour and stimulated with TGF-β2 for 48 hours. Representative phase-contrast images of the scratched explants were acquired. (D) The shortest distances between the edges of migrated cells (including their protrusions) from both sides were measured. Compared with the control, TGF-β2 stimulation caused significant cell migration 12 and 18 hours after scratching, which was attenuated by SAHA pretreatment. Scale bar denotes 500 μm. *P < 0.05 compared with control and SAHA + TGF-β2. Data are shown as means ± SE.
Figure 6
 
Effects of SAHA on cytokine concentrations. HConFs treated with or without 5 μM SAHA for 1 hour followed by TGF-β2 for 48 hours, and supernatants were collected to evaluate the cytokine concentrations affected in GO analysis using multiplex immunoassay. Among the inflammatory cytokines, IL-8 and MCP-1 were indeed significantly increased by SAHA, both in the presence and absence of TGF-β2, whereas CXCL1, CXCL6, and BMP2 were unaffected by SAHA. Although treatment with TGF-β2 alone did not significantly change IL-8 or MCP-1 levels, TGF-β2 differently affected IL-8 and MCP-1 levels in the presence of SAHA; TGF-β2 significantly increased IL-8 levels in the presence of SAHA, whereas TGF-β2 significantly decreased MCP-1 levels. On the other hand, TGF-β2 induced the angiogenic factors, PDGF-AA and VEGF, which were markedly repressed by SAHA. *P < 0.05, **P < 0.001, ***P < 0.0001. Data are shown as means ± SE.
Figure 6
 
Effects of SAHA on cytokine concentrations. HConFs treated with or without 5 μM SAHA for 1 hour followed by TGF-β2 for 48 hours, and supernatants were collected to evaluate the cytokine concentrations affected in GO analysis using multiplex immunoassay. Among the inflammatory cytokines, IL-8 and MCP-1 were indeed significantly increased by SAHA, both in the presence and absence of TGF-β2, whereas CXCL1, CXCL6, and BMP2 were unaffected by SAHA. Although treatment with TGF-β2 alone did not significantly change IL-8 or MCP-1 levels, TGF-β2 differently affected IL-8 and MCP-1 levels in the presence of SAHA; TGF-β2 significantly increased IL-8 levels in the presence of SAHA, whereas TGF-β2 significantly decreased MCP-1 levels. On the other hand, TGF-β2 induced the angiogenic factors, PDGF-AA and VEGF, which were markedly repressed by SAHA. *P < 0.05, **P < 0.001, ***P < 0.0001. Data are shown as means ± SE.
Figure 7
 
Effects of SAHA on angiogenesis. HConFs were pretreated with or without 5 μM SAHA, followed by TGF-β2 for 48 hours. Supernatants were collected, and HUVECs were seeded onto Matrigel-coated 96-well plates with each supernatant. Phase-contrast images of tube formation of HUVECs were taken after 12 hours. Supernatant of TGF-β2–treated HConFs induced tube formation of HUVECs, which was potently inhibited by SAHA pretreatment.
Figure 7
 
Effects of SAHA on angiogenesis. HConFs were pretreated with or without 5 μM SAHA, followed by TGF-β2 for 48 hours. Supernatants were collected, and HUVECs were seeded onto Matrigel-coated 96-well plates with each supernatant. Phase-contrast images of tube formation of HUVECs were taken after 12 hours. Supernatant of TGF-β2–treated HConFs induced tube formation of HUVECs, which was potently inhibited by SAHA pretreatment.
Table 1
 
Representative GO Biological Processes of Genes in SAHA-Treated Cells Up-Regulated in the Presence of TGF-β2
Table 1
 
Representative GO Biological Processes of Genes in SAHA-Treated Cells Up-Regulated in the Presence of TGF-β2
Table 2
 
Representative GO Biological Processes of Genes in SAHA-Treated Cells Down-Regulated in the Presence of TGF-β2
Table 2
 
Representative GO Biological Processes of Genes in SAHA-Treated Cells Down-Regulated in the Presence of TGF-β2
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