Table 1.
Primer sequences used in real-time PCR.
Table 2.
Antibodies used for RPE cells.
Table 3.
Antibodies used for bovine choroidal endothelial cells.
Table 4.
Antibodies used for human umbilical vein endothelial cells.
Table 5.
Primer sequences used in ChIP assay.
Table 6.
Antibodies used for immunohistochemistry.
Fig 1.
TSA induces RPE cell cycle arrest by inhibiting cell proliferation.
Cell cycle analysis of RPE cells treated with 0–1 μM TSA for 24 h, fixed with ice-cold 70% ethanol and stained with propidium iodide by an EPICS XL-MCL flow cytometer. (A) With increasing doses of TSA, significantly more RPE cells were in the G1 phase and fewer in the S phase than untreated cells. (*: t test p<0.01) (B) Percentage of cells in each phase of the cell cycle after TSA treatment. (C) PrestoBlue assay was performed on RPE cells treated with 0, 0.05, 0.1, 0.3, 0.5, 0.7 or 1 μM TSA to determine the toxicity of TSA on RPE cells. The lowest number of viable RPE cells was seen when cells were treated with 1 μM TSA for 24 h (82.0% viable cells). (*: t test p< 0.0001).
Fig 2.
TSA promotes RPE cell attachment to fibronectin.
RPE cells treated with 0–0.7 μM TSA for 24 h were allowed to attach to 96-well plates coated with fibronectin for 5, 10, 15 or 30 min. The attachment of RPE cells to fibronectin was evaluated by a modified MTT assay. Absorbance was read at 550 nm. TSA-treated RPE cells showed a (A) dose- and (B) time-dependent increase in attachment to fibronectin. (*: t test p<0.05; **: t test p<0.01).
Fig 3.
TSA inhibits PDGF-induced RPE cell migration.
RPE cells treated with 0–0.7 μM TSA for 24 h were placed on the top of a fibronectin coated-Boyden chamber, with 10 ng/mL of PDGF and DMEM with 0.4% FBS added to the bottom chamber and incubated for 5 h. Four randomly chosen fields were counted per inserts, and the results presented are averages of the four fields from each insert from three independent experiments. TSA inhibits RPE cell migration at 0.5 and 0.7 μM. (*: t test p<0.05).
Fig 4.
TSA suppresses α-SMA induced by TGF-β.
RPE cells were treated with 20 ng/mL of TGF-β only, or co-treated with 0–0.7 μM TSA and 20 ng/mL of TGF-β for 72 h. (A) The expression of α-SMA is induced by TGF-β treatment by 2-fold, and this up-regulation was reduced by TSA in a dose-dependent manner. (B) Densitometry data for Western blot result of α-SMA. (*: t test p<0.05; **: t test p<0.01).
Fig 5.
TSA reduces HIF-1α and VEGF expression and up-regulates the expression of PEDF.
Real-time PCR (A) and Western blot assays (B) were performed on RPE cells treated with 0–0.5 μM TSA and 150 μM CoCl2. (A) Cells were treated with 0–0.5 μM TSA for 14 h and then co-treated with 150 μM CoCl2 for 6 h for the analysis of gene expression by real-time PCR. Changes in HIF-1α mRNA levels were not statistically significant. CoCl2 causes a fourfold enhancement of VEGF mRNA expression; but at 0.5 μM TSA, the mRNA level of VEGF reduces to less than half of that in cells treated with CoCl2 only. TSA induces a statistically significant increase in the mRNA level of PEDF. (B) Cells were treated with 0–0.5 μM TSA for 18 h and then co-treated with 150 μM CoCl2 for 6 h for Western blot analysis. TSA reduces the CoCl2-induced HIF-1α and VEGF protein levels by 4.3-fold and 5.7-fold, respectively, and up-regulates PEDF protein level by threefold. (C) Densitometry data for Western blot of HIF-1α, VEGF and PEDF. (*: t test p<0.05; **: t test p<0.01).
Fig 6.
TSA regulates promoter activities of VEGF and PEDF.
After RPE cells were harvested, chromatin fixed with 1% formaldehyde and fragmented by sonication was immunoprecipitated with mouse IgG, anti-RNA polymerase II or anti-acetyl-histone H3 antibodies. Released chromatin was then amplified by PCR using primers targeting VEGF and PEDF encompassing the region from 200 bp upstream of the transcription start site to 200 bp downstream of the transcription start site. Amplified chromatin was then run on a 1% agarose gel. (A) For VEGF, less promoter opening was found in TSA and CoCl2-treated cells than in CoCl2-only-treated cells and untreated cells. For PEDF, more promoter opening was found in TSA and CoCl2-treated cells than in CoCl2-only-treated cells and untreated cells. (B) Densitometry of ChIP assay result normalized by input levels. (*: t test p<0.05).
Fig 7.
TSA increases apoptotic BCECs.
TUNEL assay was performed on BCECs treated with 500 μM hydrogen peroxide for 4 h, or 0.7 μM TSA for 24 h. TSA moderately increases the number of TUNEL-positive BCECs. (*: t test p<0.0002) (bars = 10 μm).
Fig 8.
TSA activates caspase 3 but inhibits the activation of Akt and p42/44 in BCECs.
(A) BCECs were treated with 0–0.7 μM TSA for 24 h. TSA activates caspase 3 at the concentrations of 0.5 and 0.7 μM, and blocks Akt phosphorylation in a dose-dependent manner. In untreated cells and cells treated with low concentrations of TSA (0.05–0.1 μM), p42/44 was activated, but the activation was completely obliterated at higher TSA concentrations (0.3–0.7 μM). (B) Densitometry data for Western blot results. (*: t test p<0.05).
Fig 9.
TSA impedes VEGF-induced tube formation in HUVECs.
1 × 104 of HUVECs treated with 0–1 μM TSA for 24 h were transferred in 150 μL of endothelium basal medium + 1% FBS with or without 25 ng/mL of human recombinant VEGF and the corresponding concentrations of TSA then incubated on 50 μL of Geltrex Reduced Growth Factor Basement Membrane Matrix gel in 96-well plates for 2 h. (A) Phase-contrast microscopy documented that tube formation induced by VEGF was inhibited by TSA in a dose-dependent manner. (B) Quantification of the amount of tube formation under different treatment conditions. (*: t test p<0.05) (bar = 200 μm).
Fig 10.
TSA inhibits VEGFR2 in BCECs and HUVECs.
Real-time PCR (A) and Western blot (B) were performed on BCECs (left panel) and HUVECs (right panel) treated with 0–0.7 μM TSA for 24h or 48 h. (A-B) TSA exerts a dose-dependent reduction effect on the mRNA and protein levels of VEGFR2. (C) Densitometry data for Western blot result of VEGFR2 normalized by GAPDH levels for BCECs (left panel) and HUVECs (right panel). (*: t test p<0.05, **: t test p<0.01) (D) HUVECs were treated with 0–0.7 μM TSA for 48 h, and then stimulated with 25 ng/mL of human recombinant VEGF for 10 min, followed by Western blot analysis (left panel). VEGF significantly induces the phosphorylation of VEGFR2, but the phosphorylation was attenuated by TSA at 0.7 μM, concomitant with a down-regulation of VEGFR2 total protein level. Densitometry data (right panel) for Western blot result of VEGFR2 and phospho-VEGFR2 normalized by GAPDH levels. (*: t test p<0.05) (E) ChIP assay was performed as described in Fig. 3 on HUVECs treated with 0.5 μM TSA for 48 h. Released chromatin was then amplified by PCR using primers targeting VEGFR2 encompassing the region from 200 bp upstream of the transcription start site to 200 bp downstream of the transcription start site. Amplified chromatin was then run on a 1% agarose gel. Less promoter opening was found in TSA-treated cells than in untreated cells. Densitometry of ChIP assay result normalized by input levels. (*: t test p<0.05).
Fig 11.
TSA reduces fluorescence leakage in laser-induced mouse CNV model.
After laser photocoagulation on day 0, C57Bl/6 mice received intra-peritoneal injections of either PBS or TSA (20 mg/kg) every 48 h for 7 or 14 days. (A, B) Angiogram pictures were taken on days 7 and 14, 3 min after a 0.1 mL of 2.5% fluorescein sodium injection. For both days 7 and 14, TSA-injected mice showed significantly less fluorescence leakage than PBS-treated mice. (*: t test p<0.05; n = 10 mice/group).
Fig 12.
Histological analysis of mouse CNV lesions after TSA treatment.
8 μm sections were stained with hematoxylin and eosin to study the histology of CNV lesions induced by laser photocoagulation in mice treated with PBS or TSA every 48 h for 7 or 14 days. (A, B) On both days 7 and 14, the area of the CNV lesions was significantly smaller in the TSA-treated mice than in mice that received only PBS. White arrows denote the location of lesions. (*: t test p<0.05; n = 8 mice in PBS group, n = 9 mice in TSA group; bars = 50 μm)
Fig 13.
CNV volume measurements after TSA treatment.
Mouse eyes were fixed with 10% formalin and eyecups were obtained by removing the anterior poles and neurosensory retina. Eyecups containing the RPE-choroid-sclera complex were blocked with PBS containing 1% BSA and 0.5% triton X-100 and then incubated with 10 μg/mL of FITC-isolectin B4 overnight at 4°C. Fluorescent images captured using the 20× objective of a scanning confocal microscope were analyzed. On both days 7 and 14, the sizes of CNV volumes were much smaller in TSA-treated mice than in mice that received PBS only. (*: t test p<0.05, **: t test p<0.005; n = 10/group) (bar = 50 μm).
Fig 14.
TSA reduces VEGF, VEGFR2 and α-SMA in mouse CNV lesions.
Immunohistochemical staining was performed on murine retinal cryostat sections in CNV lesions day 7 post-laser for (A-B) VEGF, (C-D) VEGFR2 and (E-F) α-SMA. Figures on the left panel (A, C and E) are from PBS control mice, and figures on the right panel (B, D and F) are from TSA-treated mice. TSA reduced the amount of cells stained positively for (A) VEGF, (C) VEGFR2 and (E) α-SMA, when compared to PBS controls (B, D and F). (G) Quantification of positively stained area for each protein normalized by the size of the CNV lesions. (*: t test p<0.05; **: t test p<0.01; n = 4/group; bar = 50 μm).