January 2010
Volume 51, Issue 1
Free
Biochemistry and Molecular Biology  |   January 2010
Inhibition of JAK2/STAT3-Mediated VEGF Upregulation under High Glucose Conditions by PEDF through a Mitochondrial ROS Pathway In Vitro
Author Affiliations & Notes
  • Zhi Zheng
    From the Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, China;
  • Haibing Chen
    Shanghai Clinical Center for Diabetes, Shanghai Diabetes Institute, Department of Endocrinology and Metabolism, Shanghai Jiaotong University Affiliated Sixth People's Hospital, Shanghai, China.
  • Hui Zhao
    From the Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, China;
  • Kun Liu
    From the Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, China;
  • Dawei Luo
    From the Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, China;
  • Yongdong Chen
    From the Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, China;
  • Yihui Chen
    From the Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, China;
  • Xiaolu Yang
    From the Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, China;
  • Qing Gu
    From the Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, China;
  • Xun Xu
    From the Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Shanghai, China;
  • Corresponding author: Xun Xu, Department of Ophthalmology, Shanghai First People's Hospital Affiliated Shanghai Jiao Tong University, Haining Road 100, Shanghai 200080, China; xuxun60@yahoo.com.cn
  • Footnotes
    2  These authors contributed equally to the work presented here and should therefore be regarded as equivalent authors.
Investigative Ophthalmology & Visual Science January 2010, Vol.51, 64-71. doi:10.1167/iovs.09-3511
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      Zhi Zheng, Haibing Chen, Hui Zhao, Kun Liu, Dawei Luo, Yongdong Chen, Yihui Chen, Xiaolu Yang, Qing Gu, Xun Xu; Inhibition of JAK2/STAT3-Mediated VEGF Upregulation under High Glucose Conditions by PEDF through a Mitochondrial ROS Pathway In Vitro. Invest. Ophthalmol. Vis. Sci. 2010;51(1):64-71. doi: 10.1167/iovs.09-3511.

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

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Abstract

Purpose.: Hyperglycemia-induced mitochondrial reactive oxygen species (ROS) production plays an important role in the development of complications of diabetes such as retinopathy. However, whether pigment epithelium–derived factor (PEDF) can decrease ROS production remains uncertain. The aim of this study was to clarify whether PEDF can decrease mitochondria-derived ROS generation and subsequently downregulate vascular endothelial growth factor (VEGF) expression; the authors also investigated the involvement of Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) in the process.

Methods.: Bovine retinal capillary endothelial cells (BRECs) were exposed to normal glucose (NG), H2O2, or high glucose (HG) in the presence or absence of PEDF. Expression of JAK2/STAT3, VEGF, uncoupling protein (UCP)-2, and proliferator-activated receptor gamma (PPARγ) in the BRECs was examined by Western blot analysis assay; VEGF and UCP-2 mRNA were determined by real-time RT-PCR. Mitochondrial membrane potential (Δψm) and ROS production were assayed using JC-1 and CM-H2DCFDA, respectively.

Results.: HG exposure caused hyperpolarization of Δψm and increased ROS generation in BRECs; meanwhile, like H2O2, it also induced the phosphorylation of JAK2/STAT3 and increased VEGF expression; these changes were inhibited by PEDF. The authors also found that PEDF-induced ROS inhibition was a result of decreased Δψm, which was caused by the upregulation of PPARγ and UCP-2 expression.

Conclusions.: For the first time it has been demonstrated that PEDF can decrease mitochondria-derived ROS generation and subsequently downregulate VEGF expression, possibly through inhibiting HG-induced JAK2/STAT3 activation, which may offer a promising strategy for halting the development of complications of diabetes.

Increasing evidence shows that reactive oxygen species (ROS) plays an important role in the development of complications of diabetes, such as retinopathy; hyperglycemia-induced ROS production is primarily thought to be associated with mitochondria and NADPH oxidase. 17 Recently, a unifying hypothesis has been proposed that uncoupling protein (UCP)-2–mediated mitochondrial ROS production in a chronic hyperglycemia setting may be a key initiator in the following pathogenic pathways: increased polyol pathway flux, increased production of advanced glycation end-products, activation of protein kinase C, and increased hexosamine pathway flux. 4,8 Therefore, how to inhibit mitochondria-derived ROS generation has become a focus of study in the treatment of complications of diabetes. 
Pigment epithelium–derived factor (PEDF), a glycoprotein of the serine protease inhibitor superfamily, has been identified as a retinal pigment epithelium–derived protein with neuronal differentiating activity in human retinoblastoma cells. 9 PEDF has been shown to have potent antiangiogenic activity in vitro and in vivo; it can inhibit retinal endothelial cell growth and migration and suppress ischemia-induced retinal neovascularization. 10,11 A study has demonstrated that PEDF significantly decreased VEGF expression and ROS production in diabetic retinopathy (DR), and the inhibitory effect of PEDF occurred through the suppression of NADPH oxidase-mediated ROS generation. 1215 However, whether PEDF can decrease mitochondria-derived ROS remains uncertain. 
Janus kinase (JAK) and signal transducer and activator of transcription (STAT) proteins were originally defined in the context of interferon signaling. 16 STATs have been found to be involved in such processes as innate and adaptive immune responses, embryonic development, cell differentiation, cell proliferation, survival, and apoptosis. 17,18 Furthermore, the STAT family has become a therapeutic target for cancer in humans. 19 Recent findings suggest that high glucose (HG) can activate intracellular signaling processes; these pathways can activate the JAK/STAT signaling cascades in glomerular mesangial cells and subsequently stimulate excessive proliferation and growth of glomerular mesangial cells, contributing to diabetic nephropathy. 20 In addition, it has been found that adenoviral transfection of the dominant-negative form of STAT3 abolished the leptin-induced upregulation of vascular endothelium growth factor (VEGF) in retinal endothelial cells. 21 Therefore, it is of interest to investigate the possible role of JAK2/STAT3 in the inhibition of VEGF expression by PEDF in cells exposed to HG. We demonstrated that PEDF inhibited HG-induced ROS generation through the mitochondria pathway and the NADPH oxidase pathway and subsequently downregulated VEGF expression, partly by preventing JAK2/STAT3 activation. 
Materials and Methods
The present study conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was carried out after approval was received from the local ethics committee/institutional board. All chemicals were of reagent grade and were purchased from Sigma Chemicals (St. Louis, MO), unless otherwise stated. 
Cell Culture
Bovine retinal capillary endothelial cells (BRECs) were cultured as described previously. 22 Cultured endothelial cells of 3 to 5 passages were used in the following experiments. In each case, the confluent BRECs were maintained in serum-free DMEM (0.4% bovine serum albumin). The cells were incubated either with normal d-glucose (5 mM; NG), NG (5 mM) plus l-glucose (25 mM; NG+L), NG (5 mM) plus H2O2 (500 μM), NG (5 mM) plus 100 nM PEDF (Upstate, Lake Placid, NY), or 10 mM N-acetylcysteine (NAC, a general ROS scavenger). Cells were also cultured with high d-glucose (30 mM; HG) alone or in the presence of 100 nM PEDF, 10 mM NAC, 10 μM AG490 (a JAK2 inhibitor), or 10 μM rosiglitazone (a peroxisome proliferator–activated receptor gamma (PPARγ) agonist for 24 to 48 hours. 
Antisense Oligonucleotide Treatment of JAK2, STAT3, and Uncoupling Protein-2
Synthesis of JAK2 and STAT3 antisense oligonucleotides and the following procedures were conducted as previously described. 23,24 The JAK2 antisense oligonucleotide sequence was 5′-GCTTGTGAGAAAGC-3′, and the corresponding sense oligonucleotide sequence was 5′-GTCCCTATACGAAC-3′; the STAT3 antisense oligonucleotide sequence was 5′-CCATTGGGCCATCCTGTTTCT-3′, and the corresponding sense oligonucleotide sequence was 5′-AGAAACAGGATGGCCCAATGG-3′; the UCP-2 antisense oligonucleotide sequence was 5′-TGAGATCTGCAATACA-3′, and the corresponding sense oligonucleotide sequence was 5′-TGTATTGCAGATCTCA-3′. After treatment with the antisense or sense oligonucleotide for 24 hours, the medium was removed, serum-free DMEM in NG was added, and the cells were allowed to recover for 30 minutes. BRECs were washed once with serum-free DMEM and were growth-arrested in the medium for 24 and 48 hours in either NG (5 mM) or HG (30 mM). 
Real-time RT-PCR Analysis of VEGF and UCP-2 mRNA
Total RNA was extracted from BRECs with reagent (Trizol; Invitrogen Life Technologies, Gaithersburg, MD) and was stored at −80°C. A quantitative polymerase chain reaction (qPCR) kit (DyNAmo Flash SYBR Green; Finnzymes Oy, Espoo, Finland) was used according to the manufacturer's instructions. The primer sequences (sense/antisense) used were as follows: VEGF, 5′-GCGGGCTGCTGCAATG-3′/5′-TGCAACGCGAGTCTGTGTTT-3′; UCP-2, 5′-TCTGACCATGGTGCGTACTGA-3′/5′-GACAATGGCATTACGAGCAAC-3′; β-actin, 5′-GCACCGCAAATGCTTCTA-3′/5′-GGTCTTTACGGATGTCAACG-3′. The specificity of the amplification product was determined by a melting curve analysis. Standard curves were generated for each gene by preparing serial dilutions of the respective cDNA gene template of known quantities. Relative quantities of each gene were obtained by normalizing their signals to those of β-actin. 
Western Blot Analysis of JAK2, STAT3, UCP-2, and PPARγ Proteins
Approximately 3 × 106 BRECs were collected and lysed in lysis buffer (NP40 1%, Tris 10 mM, NaCl 200 mM, EDTA 5 mM, glycerol 10%, and protease inhibitors; pH 7). Cell samples were centrifuged at 12,000 rpm for 20 minutes at 4°C, and clear supernatants were collected. The protein concentration in the supernatant was measured using the Bio-Rad (Hercules, CA) DC protein assay. Fifty micrograms of protein from each sample was subjected to SDS-PAGE using a Bio-Rad miniature slab gel apparatus and was electrophoretically transferred onto a nitrocellulose sheet. The sheet was blocked with 5% nonfat dried milk solution and incubated overnight with partially purified rabbit anti-JAK2 and anti-phospho-JAK2 polyclonal antibody (Upstate; 1:500), rabbit anti-STAT3 and mouse anti-phospho-STAT3 polyclonal antibody (Upstate; 1:500), rabbit anti-UCP-2 polyclonal antibody (Merck, Whitehouse Station, NJ; 1:500), and rabbit anti-PPARγ polyclonal antibody (Millipore, Billerica, MA; 1:500). β-Actin (monoclonal anti-β-actin; Sigma; 1:1000) expression was used as an internal control to confirm equivalent total protein loading. 
Determination of VEGF Level by ELISA
VEGF level in the supernatant was determined by ELISA with a VEGF assay kit (Quantikine; R&D Systems, Inc., Minneapolis, MN). The sensitivity of the VEGF assay was 31.2 pg/mL, and the intra-assay and interassay variations were 6.6% and 7.5%, respectively. Serial dilutions of recombinant human VEGF were included in all assays to serve as standards. 
Measurement of ROS Production
The production of ROS in the cells was assessed using the fluorescent probe 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate acetyl ester (CM-H2DCFDA; Molecular Probes, Eugene, OR). CM-H2DCFDA (λex, 488 nm and λem, 520 nm) is a cell-permeable indicator for ROS and remains nonfluorescent until acetate groups are removed by intracellular esterases and oxidation occurs in the cell. An aliquot of 200-μL cell suspension (∼105 cells/mL) was loaded on a 96-well polystyrene plate (FluoroNunc; Sigma) together with CM-H2DCFDA (10 μM) for 45 minutes at 37°C. Intracellular ROS production was calculated using an H2O2 standard curve (10–200 nmol/mL). 
Measurement of Mitochondrial Membrane Potential
5,5′,6,6′-Tetrachloro1,1′,3,3′-tetraethyl-benzimidazolylcarbocyanine iodide (JC-1; Molecular Probes) is a potentiometric dye that exhibits membrane potential-dependent loss when J-aggregates (polarized mitochondria) are converted to JC-1 monomers (depolarized mitochondria), as indicated by the fluorescence emission shift from red to green. Mitochondrial depolarization is manifested by an increase of the green/red fluorescence intensity ratio. Mitochondrial membrane potential (Δψm) measurement in BRECs was performed using flow cytometry (Coulter Epics XL; Beckman-Coulter, Fullerton, CA). as described in our previous study. 22  
NADPH Oxidase Assay
NADPH oxidase activity in the culture supernatants of BRECs was measured by luminescence assay in 50 mM phosphate buffer (pH 7.0) containing 1 mM EGTA, 150 mM sucrose, 5 μM lucigenin as the electron acceptor, and 100 μM NADPH as the substrate, according to the methods described by Griendling et al. 25  
Statistical Analysis
Experimental data were expressed as mean ± SD. Group mean was compared by one-way ANOVA using a software system (Prism 4.0; GraphPad, San Diego, CA) and a statistical software program (SPSS13.0 for Windows; SPSS, Chicago, IL). Pearson correlation tests were also performed. P < 0.05 was considered significant in all cases. 
Results
High Glucose–Induced Mitochondrial Hyperpolarization, Activation of NADPH Oxidase, and Increase of ROS Generation
As shown in Figures 1A–C, compared with the NG group, exposure of BRECs to HG caused the hyperpolarization of Δψm (Fig. 1A), the activation of NADPH oxidase (Fig. 1B), and a concomitant increase of ROS generation (Fig. 1C). Moreover, the Δψm and NADPH oxidase activity of endothelial cells increased with the increase of glucose level in the media (P < 0.01; data not shown). Pearson correlation tests indicated that ROS production was positively correlated with the Δψm (Fig. 1D) (r = 0.79; P = 0.02) and NADPH oxidase (Fig. 1E) in the presence of HG (r = 0.75; P = 0.03). 
Figure 1.
 
Mitochondrial membrane potential (Δψm), NADPH oxidase activity, and ROS generation in BRECs. (A) Δψm in BRECs cultured in NG (5 mM glucose), NG+PEDF, HG (30 mM glucose), HG+PEDF, and HG+PEDF+UCP-2 antisense, identified by the molecular probe JC-1. (B) NADPH oxidase activity in BRECs cultured in NG, NG+PEDF, HG, and HG+PEDF identified by the luminescence assay. (C) Intracellular ROS generation in BRECs cultured in NG, NG+PEDF, HG, HG+PEDF, HG+PEDF+UCP-2 antisense, HG+DPI (an NADPH oxidase inhibitor), and HG+DPI+UCP-2 antisense, identified by the fluorescent probe CM-H2DCFDA. (D) Correlation analysis between Δψm level and intracellular ROS generation in BRECs cultured in HG. (E) Correlation analysis between NADPH oxidase activity and intracellular ROS generation in BRECs cultured in HG. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (**P < 0.01 vs. NG; # P < 0.05 vs. HG; ## P < 0.01 vs. HG; $ P < 0.05 vs. HG+PEDF).
Figure 1.
 
Mitochondrial membrane potential (Δψm), NADPH oxidase activity, and ROS generation in BRECs. (A) Δψm in BRECs cultured in NG (5 mM glucose), NG+PEDF, HG (30 mM glucose), HG+PEDF, and HG+PEDF+UCP-2 antisense, identified by the molecular probe JC-1. (B) NADPH oxidase activity in BRECs cultured in NG, NG+PEDF, HG, and HG+PEDF identified by the luminescence assay. (C) Intracellular ROS generation in BRECs cultured in NG, NG+PEDF, HG, HG+PEDF, HG+PEDF+UCP-2 antisense, HG+DPI (an NADPH oxidase inhibitor), and HG+DPI+UCP-2 antisense, identified by the fluorescent probe CM-H2DCFDA. (D) Correlation analysis between Δψm level and intracellular ROS generation in BRECs cultured in HG. (E) Correlation analysis between NADPH oxidase activity and intracellular ROS generation in BRECs cultured in HG. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (**P < 0.01 vs. NG; # P < 0.05 vs. HG; ## P < 0.01 vs. HG; $ P < 0.05 vs. HG+PEDF).
Inhibition of Mitochondria-Derived ROS Generation through PPARγ-Mediated Upregulation of UCP-2 by PEDF
UCP-2 is believed to play various physiological roles, such as nonshivering thermogenesis, energy production, and redox balance. A major function of UCP-2 is that it serves as a sensor and a negative regulator of mitochondria-derived ROS production in cells with elevated glucose levels. 22,26 To explore the action of PEDF on Δ ψm in BRECs, we analyzed the expression of UCP-2 mRNA and protein. We found that hyperglycemia upregulated the expression of UCP-2 mRNA and protein and that the upregulation of UCP-2 was caused by increased ROS generation given that a general ROS scavenger NAC could block the action in the HG+NAC group. The inclusion of the NAC group was to rule out the negative feedback of ROS to UCP-2 in the presence of hyperglycemia so as to observe whether PEDF can directly upregulate UCP-2. We found that PEDF upregulated UCP-2 expression in BRECs exposed to NG or HG with or without NAC (Figs. 2A–D). Incubation with UCP-2–antisense oligonucleotide greatly enhanced hyperglycemia-induced Δψm and ROS production, blocking the inhibitory effect of PEDF on Δψm and ROS production (Figs. 1A, 1C). In addition, we found that DPI, a flavoprotein inhibitor of NADPH oxidase, inhibited hyperglycemia-induced ROS generation, and the action was weaker than that of PEDF. Moreover, there was no significant difference in ROS production between the HG+DPI group and the HG+DPI+UCP-2 antisense group (Fig. 1C). We also demonstrated that the UCP-2–antisense oligonucleotide prevented the synthesis of UCP-2 (Fig. 3). 
Figure 2.
 
Determination of UCP-2 mRNA and protein levels in BRECs. (A, B) UCP-2 mRNA in BRECs cultured in the NG, NG+PEDF, NG+NAC, and NG+NAC+PEDF (A), or in NG, HG, HG+PEDF, HG+NAC, HG+NAC+PEDF, and HG with PEDF in the presence of NAC with GW9662 (HG+NAC+PEDF+GW9662; B), was quantified by real-time RT-PCR. Results are expressed (relative to the NG values). (C, D) Representative Western blot analysis of UCP-2 protein expression in BRECs in the four groups (C) or in the six groups (D). Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by densitometry of UCP-2 in BRECs for all the four groups or six groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. NG; **P < 0.01 vs. NG; # P < 0.05 vs. HG; $ P < 0.05 vs. HG+NAC; & P < 0.05 vs. HG+NAC+PEDF).
Figure 2.
 
Determination of UCP-2 mRNA and protein levels in BRECs. (A, B) UCP-2 mRNA in BRECs cultured in the NG, NG+PEDF, NG+NAC, and NG+NAC+PEDF (A), or in NG, HG, HG+PEDF, HG+NAC, HG+NAC+PEDF, and HG with PEDF in the presence of NAC with GW9662 (HG+NAC+PEDF+GW9662; B), was quantified by real-time RT-PCR. Results are expressed (relative to the NG values). (C, D) Representative Western blot analysis of UCP-2 protein expression in BRECs in the four groups (C) or in the six groups (D). Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by densitometry of UCP-2 in BRECs for all the four groups or six groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. NG; **P < 0.01 vs. NG; # P < 0.05 vs. HG; $ P < 0.05 vs. HG+NAC; & P < 0.05 vs. HG+NAC+PEDF).
Figure 3.
 
Effect of JAK2, STAT3, and UCP-2 sense and antisense oligonucleotides on JAK2, STAT3, and UCP-2 expression in BRECs incubated in NG. BRECs were treated with JAK2, STAT3, or UCP-2 sense and antisense oligonucleotides for the times indicated and were lysed, and JAK2, STAT, or UCP-2 was immunoprecipitated from the lysates with the specific anti-JAK2, anti-STAT3, or anti-UCP-2 antibody. Precipitated proteins were then immunoblotted with the specific anti-JAK2, anti-STAT3, or anti-UCP-2 antibody. The experiments were repeated independently at least three times.
Figure 3.
 
Effect of JAK2, STAT3, and UCP-2 sense and antisense oligonucleotides on JAK2, STAT3, and UCP-2 expression in BRECs incubated in NG. BRECs were treated with JAK2, STAT3, or UCP-2 sense and antisense oligonucleotides for the times indicated and were lysed, and JAK2, STAT, or UCP-2 was immunoprecipitated from the lysates with the specific anti-JAK2, anti-STAT3, or anti-UCP-2 antibody. Precipitated proteins were then immunoblotted with the specific anti-JAK2, anti-STAT3, or anti-UCP-2 antibody. The experiments were repeated independently at least three times.
To further explore the mechanism by which PEDF induces UCP-2 upregulation, we measured the expression of PPARγ protein in BRECs after 24-hour incubation. As illustrated in Figure 4, HG increased PPARγ protein expression; meanwhile, PEDF upregulated their expression under NG or HG conditions in the BRECs (Fig. 4A), as did rosiglitazone (Fig. 4B). When PPARγ expression was inhibited by an irreversible PPARγ inhibitor, GW9662, PEDF-induced increases in UCP-2 expression were blocked, suggesting that PEDF upregulates UCP-2 expression through, at least in part, PPARγ (Figs. 2B, 2D). 
Figure 4.
 
Determination of PPARγ protein level in BRECs. (A) Representative Western blot analysis of PPARγ protein expression in BRECs in NG, NG+PEDF, HG, and HG+PEDF for 48 hours. (B) Representative Western blot analysis of PPARγ protein expression in BRECs in NG, NG+rosiglitazone (NG+R), HG, and HG+R for 48 hours. Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by PPARγ densitometry in BRECs in all four groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. NG; **P < 0.01 vs. NG; ##P < 0.01 vs. HG).
Figure 4.
 
Determination of PPARγ protein level in BRECs. (A) Representative Western blot analysis of PPARγ protein expression in BRECs in NG, NG+PEDF, HG, and HG+PEDF for 48 hours. (B) Representative Western blot analysis of PPARγ protein expression in BRECs in NG, NG+rosiglitazone (NG+R), HG, and HG+R for 48 hours. Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by PPARγ densitometry in BRECs in all four groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. NG; **P < 0.01 vs. NG; ##P < 0.01 vs. HG).
High Glucose and H2O2-Mediated Tyrosine Phosphorylation of JAK2 and STAT3
To determine whether HG can induce the tyrosine phosphorylation of JAK2 (p-JAK2) and the tyrosine phosphorylation of STAT3 (p-STAT3), BRECs were incubated for 24 and 48 hours in serum-free DMEM containing either NG, NG+L, or HG. As shown in Figure 5, under NG or NG+L conditions, there was no significant tyrosine phosphorylation of JAK2 and STAT3, suggesting that osmolarity did not affect their tyrosine phosphorylation. However, JAK2 and STAT3 were more obviously tyrosine-phosphorylated under HG conditions at both 24 and 48 hours than were those under NG or NG+L conditions, suggesting that HG promotes the activation of JAK2 and STAT3. However, there was no significant difference in the HG-induced JAK2 and STAT3 tyrosine phosphorylation between the 24-hour and 48-hour HG exposure. Lysates were also immunoblotted with an anti-JAK2 antibody or an anti-STAT3 antibody recognizing both phosphorylated and nonphosphorylated forms of JAK2 or STAT3. Equal amounts of JAK2 and STAT3 were detected under all conditions by the nonphosphospecific antibody, indicating that the differences detected with the phosphotyrosine-specific antibody were not the result of differences of total JAK2 or STAT3 protein loaded in each lane (Fig. 5). 
Figure 5.
 
Determination of JAK2, phosphorylation of JAK2 (p-JAK2), STAT3, and phosphorylation of STAT3 (p-STAT3) levels in BRECs. Representative Western blot analysis of p-JAK2, JAK2, p-STAT3, and STAT3 protein expression in BRECs incubated in serum-free medium containing NG, 5 mM NG+L, HG for 24 and 48 hours, or NG concentration with H2O2 (500 μM) for 2 and 4 hours. Cells were lysed and were immunoblotted with either phosphotyrosine-specific or nonphosphospecific anti-JAK2 antibodies, in addition to either phosphotyrosine-specific or nonphosphospecific anti-STAT3 antibodies. Quantification of signal intensity by densitometry of the four molecules in BRECs in the four groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (**P < 0.01 vs. NG or NG+L in 24 or 48 hours).
Figure 5.
 
Determination of JAK2, phosphorylation of JAK2 (p-JAK2), STAT3, and phosphorylation of STAT3 (p-STAT3) levels in BRECs. Representative Western blot analysis of p-JAK2, JAK2, p-STAT3, and STAT3 protein expression in BRECs incubated in serum-free medium containing NG, 5 mM NG+L, HG for 24 and 48 hours, or NG concentration with H2O2 (500 μM) for 2 and 4 hours. Cells were lysed and were immunoblotted with either phosphotyrosine-specific or nonphosphospecific anti-JAK2 antibodies, in addition to either phosphotyrosine-specific or nonphosphospecific anti-STAT3 antibodies. Quantification of signal intensity by densitometry of the four molecules in BRECs in the four groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (**P < 0.01 vs. NG or NG+L in 24 or 48 hours).
In addition, we also found that incubation with H2O2 resulted in the tyrosine phosphorylation of JAK2 and STAT3 in BRECs after incubation for 2 and 4 hours, with no significant difference found between the 2- and 4-hour H2O2 exposure groups (Fig. 5). The effect of H2O2 on p-JAK2/p-STAT3 was dose dependent (data not shown). Moreover, when BRECs were incubated in HG in the presence of ROS scavenger NAC, the upregulation of p-JAK2/p-STAT3 was blocked, indicating that ROS mediates JAK2/STAT3 activation (Fig. 6A). 
Figure 6.
 
Determination of JAK2, p-JAK2, STAT3, and p-STAT3 levels in BRECs. (A) Representative Western blot analysis of JAK2, p-JAK2, STAT3, and p-STAT3 in BRECs in NG, NG+NAC, NG+PEDF, HG, HG+NAC, HG+PEDF. (B) Representative Western blot analysis of p-JAK2/p-STAT3 in BRECs cultured in serum-free DMEM containing HG in the presence of PEDF (0, 10, 100, and 500 nM) for 24 hours. Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by densitometry of the four molecules in BRECs. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. 0 nM PEDF; **P < 0.01 vs. NG; ## P < 0.01 vs. HG).
Figure 6.
 
Determination of JAK2, p-JAK2, STAT3, and p-STAT3 levels in BRECs. (A) Representative Western blot analysis of JAK2, p-JAK2, STAT3, and p-STAT3 in BRECs in NG, NG+NAC, NG+PEDF, HG, HG+NAC, HG+PEDF. (B) Representative Western blot analysis of p-JAK2/p-STAT3 in BRECs cultured in serum-free DMEM containing HG in the presence of PEDF (0, 10, 100, and 500 nM) for 24 hours. Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by densitometry of the four molecules in BRECs. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. 0 nM PEDF; **P < 0.01 vs. NG; ## P < 0.01 vs. HG).
Decrease of HG-Induced VEGF Production by PEDF through Suppression of JAK2/STAT3 Activation
We found that both JAK2 and STAT3 antisense oligonucleotides directed against their translation initiation sites prevented the synthesis of JAK2 and STAT3 (Fig. 3). Incubation with HG for 48 hours significantly increased VEGF mRNA and protein expression, and PEDF significantly inhibited the increase of VEGF (Figs. 7A, 7B). To test the effects of JAK2 and STAT3 on VEGF production, we used two approaches, one with JAK2 specific inhibitor AG490 and the other with JAK2 and STAT3 antisense oligonucleotides. We found that both approaches significantly inhibited HG-induced VEGF mRNA and protein synthesis in BRECs (Figs. 7A, 7B). Furthermore, depleting STAT3 from BRECs by preincubation with STAT3 antisense oligonucleotides also significantly inhibited the HG-induced VEGF synthesis. Meanwhile, we found that PEDF had a similar effect on HG-induced VEGF synthesis, whereas PEDF had no such effect under NG conditions (Figs. 7A, 7B). As illustrated in Figure 6A, PEDF inhibited HG-mediated p-JAK2/p-STAT3 in a dose-dependent manner (Fig. 6B). It is suggested that activation of JAK2/STAT3 plays a key role in HG-induced VEGF synthesis and that the JAK2/STAT3/VEGF pathway is mediated by PEDF. 
Figure 7.
 
Determination of VEGF mRNA and protein levels in BRECs. VEGF m RNA in BRECs cultured in six-well plates with serum-free DMEM containing NG, NG+PEDF, NG+H2O2, HG, or HG in the presence of AG490, PEDF, JAK2 sense, JAK2 antisense oligonucleotides, STAT3 sense, or STAT3 antisense oligonucleotides for 48 hours. ELISA was used to determine accumulations of VEGF in the media in the 10 groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (real-time RT-PCR) or two times (ELISA) with similar results (**P < 0.01 vs. NG; ## P < 0.01 vs. HG).
Figure 7.
 
Determination of VEGF mRNA and protein levels in BRECs. VEGF m RNA in BRECs cultured in six-well plates with serum-free DMEM containing NG, NG+PEDF, NG+H2O2, HG, or HG in the presence of AG490, PEDF, JAK2 sense, JAK2 antisense oligonucleotides, STAT3 sense, or STAT3 antisense oligonucleotides for 48 hours. ELISA was used to determine accumulations of VEGF in the media in the 10 groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (real-time RT-PCR) or two times (ELISA) with similar results (**P < 0.01 vs. NG; ## P < 0.01 vs. HG).
Discussion
Our study demonstrates for the first time that PEDF can inhibit mitochondria-derived ROS generation by decreased Δψm and that decreased Δψm is caused by PPARγ-mediated upregulation of UCP-2, which subsequently prevents p-JAK2/p-STAT3 and results in the inhibition of VEGF expression. 
Recently, it has been demonstrated that leptin stimulates ischemia-induced retinal neovascularization, possibly through the upregulation of endothelial VEGF, which is mediated at least in part by the activation of STAT3. 19 Therefore, STAT3 antagonists may effectively impair angiogenesis. However, little information is available regarding approaches that can inhibit STAT3 activation-induced VEGF expression. Our study is the first to demonstrate that the action of PEDF might be used as a promising therapeutic strategy for the inhibition of STAT3. 
PEDF is a highly effective inhibitor of angiogenesis in cell cultures and animal models. It can inhibit the growth and migration of cultured endothelial cells and can potently suppress ischemia-induced retinal neovascularization. 10,11 Moreover, the action of PEDF on angiogenesis suggests its implication in a much wider range of diseases, including tumor proliferation and metastasis. 27 PEDF has also been found to be involved in the pathogenesis of PDR. Lower level PEDF and higher level VEGF may be related to the angiogenesis in DR, which leads to active PDR. 28,29 Recently, it was demonstrated that PEDF significantly decreased VEGF expression and inhibited VEGF-VEGF receptor 2 binding in DR. 12 In vitro, PEDF inhibited VEGF-induced phosphorylation of VEGFR-1. 30,31 This downregulation of VEGF expression by PEDF is confirmed in the retinas of rats with oxygen-induced retinopathy. Silencing of the PEDF gene by siRNA in Müller cells results in the significant upregulation of VEGF expression, suggesting that PEDF is an endogenous negative regulator of VEGF. 12 Furthermore, studies have demonstrated that PEDF inhibits VEGF expression through the suppression of NADPH oxidase-mediated ROS generation. 13 Similarly, the present study also found that PEDF inhibited NADPH oxidase activation and subsequently downregulated VEGF expression. 
Hyperglycemia-induced ROS appears to be produced mainly from mitochondrial sources and through the enzyme NADPH oxidase. 17 Our study demonstrated for the first time that both high glucose-derived NADPH oxidase activation and mitochondrial membrane hyperpolarization could increase ROS production, inducing p-JAK2/p-STAT3, and consequently could upregulate the expression of VEGF mRNA and protein in BRECs, which can be inhibited by PEDF. 
To explore the mechanism by which PEDF decreases mitochondrial membrane hyperpolarization, we tested the effect of PEDF on UCP-2 expression in BRECs. A major role of UCP-2 is that it serves as a sensor and a negative regulator of ROS production. UCP-2 shares its homology with UCP-1, 32 a moiety known to compensatively dissipate Δψm and to uncouple oxidative respiration. 33 We found that hyperglycemia-increased UCP-2 expression negatively regulated ROS production and that PEDF directly upregulated UCP-2 expression in BRECs exposed to NG or HG. Meanwhile, we verified that PEDF decreased Δψm through UCP-2 (Fig. 1A). 
We also investigated the mechanisms underlying the upregulation of UCP-2 by PEDF. PPARs are ligand-activated transcription factors belonging to the nuclear receptor superfamily. There are three isoforms of PPARs, namely PPARα, PPARδ (or β), and PPARγ. PPARs mediate transcriptional regulation through their central DNA-binding domain, which recognizes response elements in the promoters of specific target genes. 34 PPARγ is expressed in adipose tissue, endothelial cells, and vascular smooth muscle cells. 35 PPARγ has been extensively studied because of its role in fat metabolism and the clinical efficacy of PPARγ activators in diabetes. 3638 We found that PEDF increased PPARγ expression in BRECs incubated in NG or in HG. This result is similar to that reported by Ho et al. 39 We also found that PPARγ upregulation was involved in the modulation of UCP-2 expression (Figs. 2B, 2D), as demonstrated in the previous study by Ito. 40  
In conclusion, the effect of PEDF on hyperglycemia-induced oxidative stress and JAK2/STAT3/VEGF is through a mitochondrial pathway and an NADPH-oxidase pathway. The role of PEDF in the ROS/JAK2/STAT3/VEGF pathway may cast new light on the treatment of various angiogenesis-associated diseases, such as DR and tumors. 
Footnotes
 Supported by Research Fund for the National Nature Science Funding of China Grants 30772370, 30871204, and 30872828.
Footnotes
 Disclosure: Z. Zheng, None; H. Chen, None; H. Zhao, None; K. Liu, None; D. Luo, None; Yo. Chen, None; Yi. Chen, None; X. Yang, None; Q. Gu, None; X. Xu, None;
The authors thank Yu Danghui of the Second Military Medical University Press for his careful reading of the English language of the manuscript. 
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Figure 1.
 
Mitochondrial membrane potential (Δψm), NADPH oxidase activity, and ROS generation in BRECs. (A) Δψm in BRECs cultured in NG (5 mM glucose), NG+PEDF, HG (30 mM glucose), HG+PEDF, and HG+PEDF+UCP-2 antisense, identified by the molecular probe JC-1. (B) NADPH oxidase activity in BRECs cultured in NG, NG+PEDF, HG, and HG+PEDF identified by the luminescence assay. (C) Intracellular ROS generation in BRECs cultured in NG, NG+PEDF, HG, HG+PEDF, HG+PEDF+UCP-2 antisense, HG+DPI (an NADPH oxidase inhibitor), and HG+DPI+UCP-2 antisense, identified by the fluorescent probe CM-H2DCFDA. (D) Correlation analysis between Δψm level and intracellular ROS generation in BRECs cultured in HG. (E) Correlation analysis between NADPH oxidase activity and intracellular ROS generation in BRECs cultured in HG. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (**P < 0.01 vs. NG; # P < 0.05 vs. HG; ## P < 0.01 vs. HG; $ P < 0.05 vs. HG+PEDF).
Figure 1.
 
Mitochondrial membrane potential (Δψm), NADPH oxidase activity, and ROS generation in BRECs. (A) Δψm in BRECs cultured in NG (5 mM glucose), NG+PEDF, HG (30 mM glucose), HG+PEDF, and HG+PEDF+UCP-2 antisense, identified by the molecular probe JC-1. (B) NADPH oxidase activity in BRECs cultured in NG, NG+PEDF, HG, and HG+PEDF identified by the luminescence assay. (C) Intracellular ROS generation in BRECs cultured in NG, NG+PEDF, HG, HG+PEDF, HG+PEDF+UCP-2 antisense, HG+DPI (an NADPH oxidase inhibitor), and HG+DPI+UCP-2 antisense, identified by the fluorescent probe CM-H2DCFDA. (D) Correlation analysis between Δψm level and intracellular ROS generation in BRECs cultured in HG. (E) Correlation analysis between NADPH oxidase activity and intracellular ROS generation in BRECs cultured in HG. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (**P < 0.01 vs. NG; # P < 0.05 vs. HG; ## P < 0.01 vs. HG; $ P < 0.05 vs. HG+PEDF).
Figure 2.
 
Determination of UCP-2 mRNA and protein levels in BRECs. (A, B) UCP-2 mRNA in BRECs cultured in the NG, NG+PEDF, NG+NAC, and NG+NAC+PEDF (A), or in NG, HG, HG+PEDF, HG+NAC, HG+NAC+PEDF, and HG with PEDF in the presence of NAC with GW9662 (HG+NAC+PEDF+GW9662; B), was quantified by real-time RT-PCR. Results are expressed (relative to the NG values). (C, D) Representative Western blot analysis of UCP-2 protein expression in BRECs in the four groups (C) or in the six groups (D). Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by densitometry of UCP-2 in BRECs for all the four groups or six groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. NG; **P < 0.01 vs. NG; # P < 0.05 vs. HG; $ P < 0.05 vs. HG+NAC; & P < 0.05 vs. HG+NAC+PEDF).
Figure 2.
 
Determination of UCP-2 mRNA and protein levels in BRECs. (A, B) UCP-2 mRNA in BRECs cultured in the NG, NG+PEDF, NG+NAC, and NG+NAC+PEDF (A), or in NG, HG, HG+PEDF, HG+NAC, HG+NAC+PEDF, and HG with PEDF in the presence of NAC with GW9662 (HG+NAC+PEDF+GW9662; B), was quantified by real-time RT-PCR. Results are expressed (relative to the NG values). (C, D) Representative Western blot analysis of UCP-2 protein expression in BRECs in the four groups (C) or in the six groups (D). Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by densitometry of UCP-2 in BRECs for all the four groups or six groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. NG; **P < 0.01 vs. NG; # P < 0.05 vs. HG; $ P < 0.05 vs. HG+NAC; & P < 0.05 vs. HG+NAC+PEDF).
Figure 3.
 
Effect of JAK2, STAT3, and UCP-2 sense and antisense oligonucleotides on JAK2, STAT3, and UCP-2 expression in BRECs incubated in NG. BRECs were treated with JAK2, STAT3, or UCP-2 sense and antisense oligonucleotides for the times indicated and were lysed, and JAK2, STAT, or UCP-2 was immunoprecipitated from the lysates with the specific anti-JAK2, anti-STAT3, or anti-UCP-2 antibody. Precipitated proteins were then immunoblotted with the specific anti-JAK2, anti-STAT3, or anti-UCP-2 antibody. The experiments were repeated independently at least three times.
Figure 3.
 
Effect of JAK2, STAT3, and UCP-2 sense and antisense oligonucleotides on JAK2, STAT3, and UCP-2 expression in BRECs incubated in NG. BRECs were treated with JAK2, STAT3, or UCP-2 sense and antisense oligonucleotides for the times indicated and were lysed, and JAK2, STAT, or UCP-2 was immunoprecipitated from the lysates with the specific anti-JAK2, anti-STAT3, or anti-UCP-2 antibody. Precipitated proteins were then immunoblotted with the specific anti-JAK2, anti-STAT3, or anti-UCP-2 antibody. The experiments were repeated independently at least three times.
Figure 4.
 
Determination of PPARγ protein level in BRECs. (A) Representative Western blot analysis of PPARγ protein expression in BRECs in NG, NG+PEDF, HG, and HG+PEDF for 48 hours. (B) Representative Western blot analysis of PPARγ protein expression in BRECs in NG, NG+rosiglitazone (NG+R), HG, and HG+R for 48 hours. Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by PPARγ densitometry in BRECs in all four groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. NG; **P < 0.01 vs. NG; ##P < 0.01 vs. HG).
Figure 4.
 
Determination of PPARγ protein level in BRECs. (A) Representative Western blot analysis of PPARγ protein expression in BRECs in NG, NG+PEDF, HG, and HG+PEDF for 48 hours. (B) Representative Western blot analysis of PPARγ protein expression in BRECs in NG, NG+rosiglitazone (NG+R), HG, and HG+R for 48 hours. Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by PPARγ densitometry in BRECs in all four groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. NG; **P < 0.01 vs. NG; ##P < 0.01 vs. HG).
Figure 5.
 
Determination of JAK2, phosphorylation of JAK2 (p-JAK2), STAT3, and phosphorylation of STAT3 (p-STAT3) levels in BRECs. Representative Western blot analysis of p-JAK2, JAK2, p-STAT3, and STAT3 protein expression in BRECs incubated in serum-free medium containing NG, 5 mM NG+L, HG for 24 and 48 hours, or NG concentration with H2O2 (500 μM) for 2 and 4 hours. Cells were lysed and were immunoblotted with either phosphotyrosine-specific or nonphosphospecific anti-JAK2 antibodies, in addition to either phosphotyrosine-specific or nonphosphospecific anti-STAT3 antibodies. Quantification of signal intensity by densitometry of the four molecules in BRECs in the four groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (**P < 0.01 vs. NG or NG+L in 24 or 48 hours).
Figure 5.
 
Determination of JAK2, phosphorylation of JAK2 (p-JAK2), STAT3, and phosphorylation of STAT3 (p-STAT3) levels in BRECs. Representative Western blot analysis of p-JAK2, JAK2, p-STAT3, and STAT3 protein expression in BRECs incubated in serum-free medium containing NG, 5 mM NG+L, HG for 24 and 48 hours, or NG concentration with H2O2 (500 μM) for 2 and 4 hours. Cells were lysed and were immunoblotted with either phosphotyrosine-specific or nonphosphospecific anti-JAK2 antibodies, in addition to either phosphotyrosine-specific or nonphosphospecific anti-STAT3 antibodies. Quantification of signal intensity by densitometry of the four molecules in BRECs in the four groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (**P < 0.01 vs. NG or NG+L in 24 or 48 hours).
Figure 6.
 
Determination of JAK2, p-JAK2, STAT3, and p-STAT3 levels in BRECs. (A) Representative Western blot analysis of JAK2, p-JAK2, STAT3, and p-STAT3 in BRECs in NG, NG+NAC, NG+PEDF, HG, HG+NAC, HG+PEDF. (B) Representative Western blot analysis of p-JAK2/p-STAT3 in BRECs cultured in serum-free DMEM containing HG in the presence of PEDF (0, 10, 100, and 500 nM) for 24 hours. Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by densitometry of the four molecules in BRECs. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. 0 nM PEDF; **P < 0.01 vs. NG; ## P < 0.01 vs. HG).
Figure 6.
 
Determination of JAK2, p-JAK2, STAT3, and p-STAT3 levels in BRECs. (A) Representative Western blot analysis of JAK2, p-JAK2, STAT3, and p-STAT3 in BRECs in NG, NG+NAC, NG+PEDF, HG, HG+NAC, HG+PEDF. (B) Representative Western blot analysis of p-JAK2/p-STAT3 in BRECs cultured in serum-free DMEM containing HG in the presence of PEDF (0, 10, 100, and 500 nM) for 24 hours. Equal protein loading was confirmed with the β-actin antibody. Quantification of signal intensity by densitometry of the four molecules in BRECs. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (*P < 0.05 vs. 0 nM PEDF; **P < 0.01 vs. NG; ## P < 0.01 vs. HG).
Figure 7.
 
Determination of VEGF mRNA and protein levels in BRECs. VEGF m RNA in BRECs cultured in six-well plates with serum-free DMEM containing NG, NG+PEDF, NG+H2O2, HG, or HG in the presence of AG490, PEDF, JAK2 sense, JAK2 antisense oligonucleotides, STAT3 sense, or STAT3 antisense oligonucleotides for 48 hours. ELISA was used to determine accumulations of VEGF in the media in the 10 groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (real-time RT-PCR) or two times (ELISA) with similar results (**P < 0.01 vs. NG; ## P < 0.01 vs. HG).
Figure 7.
 
Determination of VEGF mRNA and protein levels in BRECs. VEGF m RNA in BRECs cultured in six-well plates with serum-free DMEM containing NG, NG+PEDF, NG+H2O2, HG, or HG in the presence of AG490, PEDF, JAK2 sense, JAK2 antisense oligonucleotides, STAT3 sense, or STAT3 antisense oligonucleotides for 48 hours. ELISA was used to determine accumulations of VEGF in the media in the 10 groups. Data represent the mean ± SD from nine cells per group, and the experiments were repeated independently at least three times (real-time RT-PCR) or two times (ELISA) with similar results (**P < 0.01 vs. NG; ## P < 0.01 vs. HG).
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