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Peroxynitrite increases VEGF expression in vascular endothelial cells via STAT3
Free Radical Biology & Medicine 39 (2005) 1353 – 1361 www.elsevier.com/locate/freeradbiomed
Original Contribution
Peroxynitrite increases VEGF expression in vascular endothelial cells via STAT3 Daniel H. Platta, Manuela Bartolia,b, Azza B. El-Remessya,c, Mohamed Al-Shabraweya, Tahira Lemtalsia, David Fultona,c, Ruth B. Caldwella,d,e,* a
Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USA Department of Pathology, Medical College of Georgia, Augusta, GA 30912, USA c Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912, USA d Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA e Department of Ophthalmology, Medical College of Georgia, Augusta, GA 30912, USA b
Received 25 February 2005; revised 20 June 2005; accepted 25 June 2005
Abstract Increased expression of vascular endothelial growth factor (VEGF) has been correlated with increased oxidative stress and formation of peroxynitrite in numerous disease conditions, including diabetic microangiopathy, tumor angiogenesis, and atherosclerosis. In this study we tested the hypothesis that peroxynitrite stimulates VEGF expression. Treatment of microvascular endothelial cells with exogenous peroxynitrite induced a time- and dose-dependent increase in VEGF mRNA, which peaked within 1 h of treatment at a concentration of 100 AM. The increase in VEGF mRNA was followed by a significant increase in VEGF protein. To define the molecular mechanisms involved, the effect of peroxynitrite was determined on the activation of two transcription factors known to regulate VEGF expression during hypoxia and tumor angiogenesis—signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor-1 (HIF-1). Peroxynitrite caused activation and nuclear translocation of STAT3, but not HIF-1. Moreover, transduction of endothelial cells with dominant-negative STAT3 abrogated the peroxynitrite-induced increase in VEGF mRNA. The increase in VEGF mRNA was also blocked by inhibitors of transcription and was unaffected by the inhibition of protein synthesis. These results indicate that peroxynitrite causes increased expression of VEGF in vascular endothelial cells by a process that requires the activation of STAT3. D 2005 Elsevier Inc. All rights reserved. Keywords: Vascular endothelial growth factor; Oxidative stress; Peroxynitrite; STAT3; HIF-1; Vascular endothelial cells; Smooth muscle cells; Free radicals
Enhanced generation of peroxynitrite (ONOO ), formed S by the diffusion-limited reaction of O2 and nitric oxide S( NO), is a hallmark of inflammatory disease conditions [1]. Whereas the cytotoxic actions of ONOO have been well described, less is known about its specific actions in modulating intracellular signaling pathways that regulate
Abbreviations: VEGF, vascular endothelial growth factor; ROS, reactive oxygen species; ONOO , peroxynitrite; NO, nitric oxide; O2 , superoxide anion; STAT3, signal transducer and activator of transcription 3; HIF-1, hypoxia inducible factor 1; CMV, cytomegalovirus. * Corresponding author. Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USA. Fax: +1 706 721 9799. E-mail address: [email protected] (R.B. Caldwell).
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0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.06.015
inflammatory responses, including induction of the angiogenic cytokine and vascular permeability factor vascular endothelial growth factor (VEGF). In particular, studies in streptozotocin-induced diabetic rat retinas have shown that scavenging ONOO or inhibiting nitric oxide synthase activity prevents diabetes-induced nitrotyrosine formation and blocks the effects of diabetes in stimulating VEGF overexpression and breakdown of the blood –retinal barrier, suggesting a causal link between ONOO formation and increases in VEGF expression [2]. Up-regulated expression of VEGF has a key role in promoting growth of dysfunctional vessels during diabetic microangiopathy, atherosclerosis, and tumor angiogenesis [3 –7]. Each of these conditions has been shown to induce
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the generation of reactive oxygen species (ROS) [8– 10]. ROS have been implicated in triggering increases in the expression of VEGF in many cell types [11– 13], but the molecular mechanisms of this effect remain to be elucidated. Studies in patients and animals and in in vitro disease models indicate that the formation of 3-nitrotyrosine, a suggested marker for ONOO , is associated with increased expression of VEGF during diabetic microvascular disease, atherosclerosis, and tumor angiogenesis [2,4,9,14 – 16]. Moreover, studies using a cultured mast cell line showed that exogenous ONOO stimulates an increase in VEGF mRNA expression [17]. The aim of this study was to determine the specific effects of ONOO on the expression of VEGF in vascular endothelial cells. Here we show that ONOO induces increased expression of VEGF via activation of the latent transcription factor STAT3.
OR, USA) according to the manufacturer’s instructions. Ten fields per plate were viewed and living and dead cells were counted as determined by staining with calcein AM (live) and ethidium homodimer-1 (dead). The results of this analysis showed that cell viability in all treatment groups was not significantly different from that in the untreated controls (96 T 2% viable cells). This experiment was repeated three times with independent batches of endothelial cells. To test whether the peroxynitrite effect on VEGF mRNA involves a transcriptional event the cultures were pretreated with actinomycin D (3 h, 4 Ag/ml) or 5,6-dichlorobenzimidazole riboside (3 h, 50 Ag/ml) (Sigma, St. Louis, MO, USA). To determine whether the peroxynitrite effect on VEGF mRNA requires an increase in protein synthesis, the cultures were pretreated with cycloheximide (Sigma) (1 h, 30 AM). Cells were then treated with the maximum effective dose of peroxynitrite (100 AM).
Methods and materials Quantitative real-time PCR analysis Cell culture Primary cultures of bovine microvascular endothelial cells (passage 7 –9) were used in these experiments [18,19]. The ONOO treatment was done on serum-starved cultures as described previously [20], with modifications. Briefly, cultures were rinsed in Hanks’ buffered salt solution (HBSS), pH 7.4. HBSS (1.95 ml) was added to each plate and 50 Al concentrated ONOO (Upstate Biotechnology, Lake Placid, NY, USA) diluted to the appropriate concentration in 0.1 N NaOH was rapidly added to the plates while mixing. Cells were incubated in the ONOO -treated buffer for 2 min at 37-C, washed with serum-free medium, and incubated at 37-C for the times indicated. The same volumes of 0.1 N NaOH or decomposed ONOO were used as controls. These control treatments did not alter any of the parameters measured. ONOO concentration was determined by spectrophotometer as described by Zou et al. [21]. In order to evaluate the relative intracellular levels of ONOO reached under these treatment conditions, formation of nitrotyrosine was determined in the ONOO -treated cultures using slot-blot techniques as described previously [20,22]. This analysis showed that the levels of nitrotyrosine formed in cultures treated with 100 –1000 AM ONOO were equivalent to those seen with 2.2 –4.8 mg/ml nitrated BSA. The relative amount of nitrotyrosine formation induced by treatment with 100 AM ONOO was roughly comparable to the levels seen in our previous studies of endothelial cells treated with high glucose or hyperoxia, which were equivalent to 1.8 and 1.6 mg/ml nitrated BSA, respectively [20,22]. To rule out possible ONOO -induced cytotoxic effects, three experiments were conducted using confluent cultures treated with varying doses of ONOO for 6, 12, and 24 h. Cell viability was determined using the Live/Dead Viability/ Cytotoxicity Assay Kit (Molecular Probes, Inc., Eugene,
VEGF mRNA expression was analyzed by quantitative RT-PCR, which was done by using the Cepheid Smart Cycler (Sunnyvale, CA, USA) with a protocol optimized for our primers as previously described [23]. Primers were designed (with the online MIT resource Primer 3 technology) to generate a PCR product of 138 bp and to include a target sequence within exons 3 and 4 of the bovine VEGF gene (Accession No. M32976; Left, 5V-ATTTTCAAGCCGTCCTGTGT-3V, and Right, 5V-TATGTGCTGGCTTTGGTGAG-3V). This sequence recognizes all isoforms of VEGFA and prevents the amplification of multiple products of differing lengths. The housekeeping gene bovine acidic ribosomal protein 1 (ARP-1) (Accession No. AF013214; Left, 5V-TACACCTTCCCACTTGCTGA-3V, and Right, 5VCTCCGACTCCTCCTTTGCTT-3V) was used as an internal standard. Nuclear fractionation To analyze the nuclear translocation of activated STAT3 and Hif-1a, nuclei were isolated from total cell lysates on a 350 mM sucrose gradient as previously described [18,19]. From the nuclear extracts, 25 Ag of protein from each sample was subjected to SDS – PAGE and immunoblotted using anti-STAT3 and anti-Hif-1a antibodies. Immunoblotting analysis Proteins were isolated and quantified as previously described [18]. VEGF was isolated by diluting 100 Ag protein to a volume of 1 ml using 10 mM Tris (pH 7.4) and 100 mM NaCl and incubating with 50 Al of equilibrated heparin –agarose beads (Sigma) as described by Ferrara and Henzel [3] and Hossain et al. [24]. Fifty micrograms of proteins or heparin –agarose-isolated VEGF was electro-
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phoresed on 10% SDS – polyacrylamide or 4– 20% Tris – HCl gradient gels (Bio-Rad Laboratories, Hercules, CA, USA) and then transferred to nitrocellulose membranes and detected with anti-human VEGF165 (Novus Biologicals, Littleton, CO, USA), anti-STAT3, anti-phospho-STAT3 (Cell Signaling Technology, Beverly, CA, USA), or antiHif-1a antibodies (BD-Transduction Labs, San Diego, CA, USA) followed by ECL chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). ELISA Confluent cultures were incubated in the presence or absence of 100 AM peroxynitrite, degraded peroxynitrite, or control medium for 12 h. Proteins were extracted from and quantified according to our established protocols [18]. Protein samples (50 Ag) were added to wells of a 96-well plate coated with VEGF antibody, incubated at 4-C overnight, and processed according to the manufacturer’s instructions (RayBiotech, Inc., Atlanta, GA, USA). Absorbance was measured in a plate reader at 450 nm. VEGF levels were calculated from absorbance values by comparison with a standard curve prepared by a six-step serial dilution of recombinant VEGF ranging from 8 to 6000 pg/ml. This experiment was repeated twice.
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quantified by measuring the distance between the wound edges before and after injury at five distinct positions (every 5 mm) using computer-assisted microscopy. Adenoviral vectors Replication-deficient adenoviruses expressing a STAT3 DNA binding domain mutant, under the control of the cytomegalovirus (CMV) promoter, were generated using the pAdTrack-CMV vector and AdEasy System [26]. The STAT3 mutant plasmid, pEF-HAStat3D, was a generous gift from Drs. Hirano and Ishihara, Osaka University Medical School, Japan [27]. Competent Escherichia coli were transformed with the STAT3D mutant and the plasmid DNA was recovered by miniprep using the Qiagen QIAprep Spin Miniprep Kit. Plasmid DNA was cut with SalI and NotI restriction endonucleases (New England Biolabs, Inc., Beverly, MA, USA) and the 2500-bp STAT3D was subcloned into the pAdTrack-CMV vector. The pAdTrack vector was electroporated into competent E. coli containing the pAdEasy vector for homologous recombination. After amplification and recovery of the viral vector, viruses were amplified in HEK293 cells, purified using a CsCl gradient, and titered by OD. Statistical analysis
Immunofluorescence Cultures were fixed with 4% paraformaldehyde and then reacted with anti-VEGF antibody followed by Oregon greenlabeled secondary antibody (Molecular Probes). Data were analyzed using the MetaMorph morphometric program (Universal Imaging Corp., West Chester, PA, USA) and fluorescence microscopy to quantify intensity of immunostaining. Specificity of the immunoreaction for VEGF was verified by control studies showing the absence of immunolabeling when the primary antibody was omitted or preadsorbed with the immunizing VEGF peptide [N-terminal sequence (aa 1 –20) of human VEGF]. Cell migration assay To evaluate the potential effects of peroxynitrite-induced increases in VEGF expression, a scratch-wound assay was used to determine the effects of peroxynitrite treatment on endothelial cell migration as described by Dimmeler et al. [25]. Briefly, ‘‘scratch’’ wounds were created by scraping cell monolayers with a precut rubber policeman to produce a wound 2 mm wide and then the wounded cultures were treated with exogenous VEGF (30 ng/ml) or concentrated conditioned medium (0.2 ml) prepared from endothelial cell cultures in 100-mm dishes which had been treated 24 h earlier with either ONOO or decomposed ONOO . The wounded cultures were photographed at specific locations immediately after wounding and 24 h later. Endothelial cell migration from the edge of the injured monolayer was
The results are expressed as the means T SE. Differences between experimental groups were evaluated by ANOVA, and the significance of the differences was determined using the Tukey test for pair-wise comparisons. Significance was defined as p < 0.05.
Results Peroxynitrite stimulation of VEGF expression To determine the effects of ONOO on VEGF expression, serum-starved endothelial cells were treated with varying doses of ONOO for different times and VEGF mRNA was quantified by real-time PCR. The ONOO treatment induced a dose-dependent increase in VEGF mRNA (Fig. 1A), beginning with 50 AM ONOO and reaching a maximum with 100 AM ONOO . This effect was also time-dependent, reaching a maximum within 1 h after treatment and declining to basal levels within 8 h (Fig. 1B). In order to test whether the ONOO -induced increase in VEGF mRNA formation resulted in an increase in VEGF protein, endothelial cells were treated with peroxynitrite (100 AM) and VEGF protein content was analyzed using immunofluorescence, Western blotting, and ELISA techniques. Densitometric analysis of the immunofluorescence and immunoblotting results showed a significant increase in VEGF protein expression in the ONOO -treated cells vs control (Fig. 2). Treatment with degraded ONOO had no
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growth factors. STAT3 is an important regulator of VEGF in angiogenesis [28,29]. Recently, we showed that VEGF autocrine expression in microvascular endothelial cells is regulated by STAT3 [23]. It has also been reported that ROS and drugs that induce oxidative stress can activate STAT3 [30]. To determine if STAT3 is activated by ONOO , we treated endothelial cells with ONOO and determined the effect on phosphorylation of STAT3 at tyrosine 705, which is required for STAT3 activation [31]. These experiments showed that ONOO (100 AM) induced a significant increase in STAT3 tyrosine phosphorylation within 5 min (Fig. 4A). Analyses with increasing concentrations of ONOO also showed a dose-dependent increase in STAT3 tyrosine phosphorylation (data not shown). When STAT proteins are activated by tyrosine phosphorylation they dimerize via their SH2 domains and are shuttled to the nucleus for transcriptional activation of target sequences [32]. To test the effects of ONOO on nuclear translocation of STAT3, we isolated nuclear proteins from treated cells and determined nuclear levels of STAT3 by Western blotting. These experiments showed that ONOO induced increased nuclear levels of STAT3 within 5 min after stimulation (Fig. 4B). Fig. 1. Effects of peroxynitrite on VEGF mRNA. Cells were (A) treated with 0 – 500 AM ONOO and allowed to incubate for 1 h after treatment or (B) treated with 100 AM ONOO and incubated 0 – 8 h after treatment, and effects on VEGF mRNA levels were determined by quantitative real-time PCR. The results are expressed as the ratio VEGF mRNA to ARP-1 mRNA T SE for three separate experiments. *p < 0.05 compared with control values.
effect on VEGF protein levels in either assay. Quantitation of VEGF protein levels using ELISA confirmed a significant (3.8-fold, p < 0.001) increase in VEGF protein formation in the ONOO -treated cells. The VEGF level in cells treated with ONOO (100 mM, 12 h) was 96 T 11 pg/ ml compared with 25 T 9 pg/ml in the untreated control cells and 17 T 5 pg/ml in the cells treated with decomposed ONOO . In order to evaluate the potential biological effects of the peroxynitrite-induced increases in VEGF protein expression, experiments were performed to determine the functional effects of ONOO treatment on endothelial cell migration. The results of experiments using a scratch-wound cell migration assay showed that medium conditioned by ONOO -treated cultures caused a significant increase in cell migration compared with medium from control cultures treated with decomposed ONOO (Fig. 3). This effect of ONOO was roughly comparable with that of exogenous VEGF. Activation of STAT3 by peroxynitrite STAT proteins are a class of latent cytoplasmic transcription factors that regulate the expression of genes involved in cellular growth induced by cytokines and
Fig. 2. Effects of ONOO on VEGF protein expression. Microvascular endothelial cells were treated with or without ONOO or degraded ONOO (100 AM) and incubated for 12 h in serum-free medium and effects on levels of VEGF protein were determined by (A) immunocytochemistry or (B) Western blotting. Results are expressed as relative optical density means T SE. n = 5 in A, n = 3 in B, *p < 0.05 compared with control values.
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Fig. 3. Effects of ONOO on cell migration. Cultures of microvascular endothelial cells were wounded as described under Methods and materials and treated with VEGF (30 ng/ml) or medium conditioned by cultures treated with or without ONOO (PN) or degraded ONOO (dPN) (100 AM). (A) Cultures were photographed immediately after wounding and 24 h later. (B) Migration was quantified by measuring the distance between the wound edges before and after treatment. The results are expressed as the % of wound closure T SE for three separate experiments. *p < 0.05 compared with control values.
Effects of peroxynitrite on hypoxia-inducible factor-1 (HIF-1) activation We next examined the potential role of HIF-1 in ONOO induced VEGF transcription. HIF-1 is a transcription factor that regulates a number of genes under conditions of low oxygen tension, including VEGF. To be activated, Hif-1a, the inducible subunit of HIF-1, must undergo cytosolic stabilization and accumulation, which is followed by nuclear translocation. It is generally thought that this stabilization does not occur in the absence of hypoxia and that Hif-1a is degraded. However, recent studies have shown that formation of reactive oxygen and nitrogen species plays a role in the activation of Hif-1a [33]. Therefore, to determine the effects of ONOO on the regulation of Hif-1a, endothelial cells were treated with 100 AM ONOO for 0, 5, 15, 30, and 60 min. Hif-1a protein was not detected in the total cell lysates (Fig. 5A), indicating that ONOO did not stabilize or
increase cytosolic levels of Hif-1a protein. To activate transcription, HIF-1 must be translocated to the nucleus and bind the hypoxia response element in the promoter of its target genes [34]. To determine if ONOO induces nuclear translocation of HIF-1, nuclear proteins isolated from treated cells and levels of Hif-1a within the nucleus were analyzed by immunoblotting. As seen in Fig. 5B, Hif-1a was not detected in the nucleus of the ONOO -treated cells. Requirement of STAT3 activity for peroxynitrite-mediated VEGF expression The above results suggest that STAT3 and not HIF-1 is involved in ONOO -mediated activation of VEGF expression. To test whether STAT3 function is required for the ONOO effect, a STAT3 DNA binding domain mutant from pEF-HAStat3D was subcloned into the pAdTrack-CMV vector in order to produce a dominant-negative adenovirus
Fig. 4. Effects of ONOO on activation and nuclear translocation of STAT3. (A) Microvascular endothelial cells were treated with ONOO (100 AM) and STAT3 activation was determined after 0 – 60 min by Western blotting of total cell lysates using an antibody against phosphotyrosine (705) STAT3. Blots were reprobed with STAT3 antibody to demonstrate equal loading. (B) Nuclear translocation of STAT3 was determined by Western blotting of isolated nuclear proteins using an antibody against STAT3. Results were quantified by densitometry and results are expressed as means T SE for three separate experiments. *p < 0.05 compared with untreated controls.
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The ONOO effect was significantly blunted in cells transduced with AdSTAT3D. The levels of VEGF mRNA in cells transduced with AdSTAT3D but not treated with ONOO were no different from that in the untreated AdGFP-transduced control cells. Effects of inhibitors of transcription on peroxynitrite-mediated VEGF expression Fig. 5. Effects of ONOO on HIF-1 activation and nuclear translocation. (A) Microvascular endothelial cells were incubated with ONOO (100 AM) and HIF-1 activation was assayed after 0 – 60 min by Western analysis of Hif-1a protein levels in total cell lysates. (B) Nuclear translocation of HIF-1 was determined by Western analysis of isolated nuclear proteins with Hif1a antibody. As a positive control (C+) for HIF-1 activation, cells were treated with cobalt chloride (150 AM) for 4 h. All blots are representative of three separate experiments.
(AdSTAT3D). The pAdTrack-CMV vector drives expression of green florescent protein (GFP), which allows tracking of cell transduction levels. Endothelial cells were transduced with the adenovirus expressing GFP only or AdSTAT3D at an estimated multiplicity of infection (m.o.i.) of 30 viral particles per cell. GFP expression was tracked for 12 h at which time an estimated 95% or more of the cells were expressing GFP (data not shown). Overexpression of dominant-negative STAT3 was verified by immunoblotting analysis using anti-HA and anti-STAT3 antibodies (data not shown). The cells were switched to serum-free medium for 12 h and then treated with or without ONOO (100 AM, 1 h) and the effects on VEGF mRNA were determined by quantitative real-time PCR. These experiments showed a significant increase in VEGF mRNA expression after ONOO treatment of cells expressing only GFP (Fig. 6).
Fig. 6. Effects of dominant-negative STAT3 on ONOO -induced VEGF expression. Microvascular endothelial cells were transduced with adenoviruses expressing STAT3 with a DNA binding domain mutation at an m.o.i. of 30. Cells were transduced with adenovirus expressing only GFP or GFP and STAT3D (DNA binding domain mutant) and treated with or without ONOO (100 AM) and incubated in serum-free medium for 1 h. VEGF mRNA levels were determined by quantitative real-time PCR. Results are expressed as the ratio of VEGF mRNA to ARP-1 mRNA T SE for three separate experiments. *p < 0.002 compared with control values. #p < 0.05 compared with ONOO -treated AdGFP.
VEGF expression is a complex process that requires transcriptional and posttranscriptional regulation [35,36]. In order to evaluate whether ONOO -induced increases in activation of STAT3 and induction of VEGF mRNA formation require increased transcription, endothelial cells were treated with the maximum effective dose of peroxynitrite (100 AM) in the presence or absence of two different inhibitors of transcription, actinomycin D and 5,6-dichlorobenzimidazole riboside (DRB). Actinomycin D specifically inhibits RNA polymerase through complex formation with deoxyguanosine residues in DNA primers. DRB inhibits transcription by blocking the activity of casein kinase II, which is required for activity of RNA polymerase II. The ONOO -induced increases in VEGF mRNA were completely inhibited in the cells treated with either actinomycin D or DRB (Fig. 7). This suggests that ONOO induced increases in VEGF required mRNA transcription. By contrast, treatment of the cultures with the protein synthesis inhibitor cycloheximide had no significant effect on the action of ONOO in increasing VEGF mRNA, indicating that the increase in VEGF mRNA does not require synthesis of new proteins.
Fig. 7. Effects of inhibitors of transcription and protein synthesis on ONOO induced VEGF mRNA. Microvascular endothelial cells were treated with or without ONOO (PN, 100 AM) and incubated for 1 h in the presence or absence of transcriptional inhibitors actinomycin D (Act-D, 4 Ag/ml) and DRB (50 Ag/ml) and the protein synthesis inhibitor cycloheximide (CHX, 30 AM). Cells were preincubated with the transcriptional inhibitors for 3 h before peroxynitrite treatment and with the protein synthesis inhibitor for 1 h. Realtime, quantitative PCR was used to measure levels of VEGF mRNA. Results are expressed as the ratio of VEGF mRNA to ARP-1 mRNA T SE for four separate experiments for actinomycin D and three separate experiments for DRB and CHX. *p < 0.001 compared with control (lane 1) values and #p < 0.001 compared with peroxynitrite (PN) alone.
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Discussion Increased expression of VEGF has been implicated in a variety of disease conditions characterized by pathological vascular growth, including diabetic microvascular disease, atherosclerosis, and tumor angiogenesis. Overexpression of VEGF has been correlated with increased levels of oxidative stress and with formation of the ONOO biomarker nitrotyrosine [2,4,9,14 –16]. In this study we tested the hypothesis that ONOO has a direct effect in stimulating the expression of VEGF in vascular endothelial cells. Here we show that treatment of primary vascular endothelial cells with exogenous ONOO induces a dose- and time-dependent increase in VEGF mRNA which is followed by increases in VEGF protein formation. Moreover, medium conditioned by cells treated with ONOO causes increases in endothelial cell migration, indicating that the peroxynitrite effect is functionally relevant. These data, together with previous work showing that ONOO is up-regulated in diabetes and that treatments which reduce ONOO formation prevent VEGF overexpression [2], strongly support the hypothesis that ONOO directly stimulates VEGF expression. The maximum increase in VEGF expression occurred with ONOO treatment at a concentration of 100 AM. Although this concentration exceeds that which is likely to occur intracellularly under either physiological or pathophysiological conditions, this is not surprising because the half-life of ONOO at pH 7.4 is on the order of seconds [37]. Both exposure time and concentration of exogenous ONOO are critical determinants for mimicking the effects of endogenous ONOO . Thus, much higher concentrations of exogenous ONOO may be needed to achieve biological responses similar to those seen with endogenous ONOO , which is continuously produced at low concentrations. To understand the molecular mechanisms of ONOO -induced VEGF expression we analyzed the activation of two regulators of VEGF expression: the transcription factors STAT3 and HIF-1. Our data demonstrated that ONOO induces the activation of STAT3 and that STAT3 activation is required for the effects of ONOO in stimulating VEGF expression. The STAT protein family of latent cytoplasmic transcription factors was originally thought to provide selective signaling because each member was activated by a different cytokine receptor. However, STAT3 is now known to be activated by a number of cytokines, growth factors, and oncogenes and to participate in different signal pathways in different cell types [38]. We have recently shown that STAT3 activation plays an important role in autocrine VEGF expression in microvascular endothelial cells [23]. Others have shown that STAT3 is activated in a wide variety of cancers and that its activation increases both VEGF expression and tumor angiogenesis [39]. Our present findings suggest that STAT3 also has a key role in ONOO -mediated induction of VEGF expression in microvascular endothelial cells. Unpublished
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studies using primary cultures of aortic smooth muscle cells indicate that ONOO treatment induces similar patterns of STAT3 activation and VEGF mRNA increases, suggesting that these effects of ONOO also apply to other vascular cell types (D.H. Platt, M. Bartoli, and R. Caldwell, unpublished results). Recent studies have shown that oxidative stress can activate STAT3 by phosphorylation of tyrosine 705 within the activation domain. Furthermore, studies of the effects of oxidative stress on tyrosine kinase activity show that receptor and nonreceptor tyrosine kinases, which can directly interact with and activate STAT3, can also be activated by ONOO [40]. Here we have shown that ONOO causes STAT3 activation as indicated by its tyrosine phosphorylation and nuclear translocation and that overexpression of dominant-negative STAT3 inhibits ONOO induced VEGF expression. These data suggest that ONOO mediated activation of STAT3 could have a key role in triggering the up-regulation of VEGF expression in diabetic microangiopathy. Studies are now in progress using animal and tissue culture models of diabetes to test this hypothesis. Our experiments showed that the ONOO treatment had no effect on either the cytosolic stability or the nuclear translocation of HIF-1, indicating that activation of HIF-1 probably does not play a role in the ONOO -induced increase in expression of VEGF. Low oxygen tension is known to stimulate cells to express VEGF by inducing activation of HIF-1. However, recent studies indicate that reactive oxygen or reactive nitrogen species can also activate HIF-1 [33,41]. These previous studies found that, S S whereas either O2 or NO alone could induce stabilization of Hif-1a, their contemporaneous production, which could favor ONOO formation, failed to induce HIF-1 activation. This is consistent with our present results. Studies have shown that hypoxia-induced increases in VEGF expression are due in large part to increases in the stability of VEGF mRNA [42]. Hydrogen peroxide- or superoxide-induced increases in VEGF mRNA levels in retinal pigment epithelial cells have been shown to be mediated exclusively by increases in mRNA stability [43]. Further investigation will be needed to determine whether ONOO increases VEGF mRNA by increasing transcriptional activity and/or by enhancing mRNA stability. However, our finding that inhibitors of transcription totally blocked the ONOO effect, whereas an inhibitor of protein synthesis was without effect, implies that transcriptional activation is likely to be involved in the process. In conclusion, ONOO has been implicated in vascular dysfunction in cardiovascular disease and the ONOO biomarker nitrotyrosine has been observed in both diabetes and atherosclerosis. Overexpression of VEGF and increased oxidative stress also occur under both conditions and are correlated with disease progression. Previous studies in models of tumor angiogenesis have shown that ONOO formation is correlated with increases in VEGF expression and that treatment of a mast cell tissue culture line with
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exogenous ONOO is associated with increases in VEGF mRNA [14,17]. Our study is the first to show that ONOO can directly stimulate VEGF expression in microvascular endothelial cells. We also show that ONOO -induced VEGF expression is mediated by activation of the transcription factor STAT3, suggesting a specific molecular role for STAT3 in VEGF overexpression and disease progression in vascular diseases characterized by increased oxidative stress.
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Acknowledgments This work was supported in part by NIH Grants R01NEI-04618 and R01-NEI-11766 and American Heart Association Grant 0365181B.
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Original Contribution
Peroxynitrite increases VEGF expression in vascular endothelial cells via STAT3 Daniel H. Platta, Manuela Bartolia,b, Azza B. El-Remessya,c, Mohamed Al-Shabraweya, Tahira Lemtalsia, David Fultona,c, Ruth B. Caldwella,d,e,* a
Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USA Department of Pathology, Medical College of Georgia, Augusta, GA 30912, USA c Department of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912, USA d Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA e Department of Ophthalmology, Medical College of Georgia, Augusta, GA 30912, USA b
Received 25 February 2005; revised 20 June 2005; accepted 25 June 2005
Abstract Increased expression of vascular endothelial growth factor (VEGF) has been correlated with increased oxidative stress and formation of peroxynitrite in numerous disease conditions, including diabetic microangiopathy, tumor angiogenesis, and atherosclerosis. In this study we tested the hypothesis that peroxynitrite stimulates VEGF expression. Treatment of microvascular endothelial cells with exogenous peroxynitrite induced a time- and dose-dependent increase in VEGF mRNA, which peaked within 1 h of treatment at a concentration of 100 AM. The increase in VEGF mRNA was followed by a significant increase in VEGF protein. To define the molecular mechanisms involved, the effect of peroxynitrite was determined on the activation of two transcription factors known to regulate VEGF expression during hypoxia and tumor angiogenesis—signal transducer and activator of transcription 3 (STAT3) and hypoxia-inducible factor-1 (HIF-1). Peroxynitrite caused activation and nuclear translocation of STAT3, but not HIF-1. Moreover, transduction of endothelial cells with dominant-negative STAT3 abrogated the peroxynitrite-induced increase in VEGF mRNA. The increase in VEGF mRNA was also blocked by inhibitors of transcription and was unaffected by the inhibition of protein synthesis. These results indicate that peroxynitrite causes increased expression of VEGF in vascular endothelial cells by a process that requires the activation of STAT3. D 2005 Elsevier Inc. All rights reserved. Keywords: Vascular endothelial growth factor; Oxidative stress; Peroxynitrite; STAT3; HIF-1; Vascular endothelial cells; Smooth muscle cells; Free radicals
Enhanced generation of peroxynitrite (ONOO ), formed S by the diffusion-limited reaction of O2 and nitric oxide S( NO), is a hallmark of inflammatory disease conditions [1]. Whereas the cytotoxic actions of ONOO have been well described, less is known about its specific actions in modulating intracellular signaling pathways that regulate
Abbreviations: VEGF, vascular endothelial growth factor; ROS, reactive oxygen species; ONOO , peroxynitrite; NO, nitric oxide; O2 , superoxide anion; STAT3, signal transducer and activator of transcription 3; HIF-1, hypoxia inducible factor 1; CMV, cytomegalovirus. * Corresponding author. Vascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USA. Fax: +1 706 721 9799. E-mail address: [email protected] (R.B. Caldwell).
S
S
0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2005.06.015
inflammatory responses, including induction of the angiogenic cytokine and vascular permeability factor vascular endothelial growth factor (VEGF). In particular, studies in streptozotocin-induced diabetic rat retinas have shown that scavenging ONOO or inhibiting nitric oxide synthase activity prevents diabetes-induced nitrotyrosine formation and blocks the effects of diabetes in stimulating VEGF overexpression and breakdown of the blood –retinal barrier, suggesting a causal link between ONOO formation and increases in VEGF expression [2]. Up-regulated expression of VEGF has a key role in promoting growth of dysfunctional vessels during diabetic microangiopathy, atherosclerosis, and tumor angiogenesis [3 –7]. Each of these conditions has been shown to induce
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the generation of reactive oxygen species (ROS) [8– 10]. ROS have been implicated in triggering increases in the expression of VEGF in many cell types [11– 13], but the molecular mechanisms of this effect remain to be elucidated. Studies in patients and animals and in in vitro disease models indicate that the formation of 3-nitrotyrosine, a suggested marker for ONOO , is associated with increased expression of VEGF during diabetic microvascular disease, atherosclerosis, and tumor angiogenesis [2,4,9,14 – 16]. Moreover, studies using a cultured mast cell line showed that exogenous ONOO stimulates an increase in VEGF mRNA expression [17]. The aim of this study was to determine the specific effects of ONOO on the expression of VEGF in vascular endothelial cells. Here we show that ONOO induces increased expression of VEGF via activation of the latent transcription factor STAT3.
OR, USA) according to the manufacturer’s instructions. Ten fields per plate were viewed and living and dead cells were counted as determined by staining with calcein AM (live) and ethidium homodimer-1 (dead). The results of this analysis showed that cell viability in all treatment groups was not significantly different from that in the untreated controls (96 T 2% viable cells). This experiment was repeated three times with independent batches of endothelial cells. To test whether the peroxynitrite effect on VEGF mRNA involves a transcriptional event the cultures were pretreated with actinomycin D (3 h, 4 Ag/ml) or 5,6-dichlorobenzimidazole riboside (3 h, 50 Ag/ml) (Sigma, St. Louis, MO, USA). To determine whether the peroxynitrite effect on VEGF mRNA requires an increase in protein synthesis, the cultures were pretreated with cycloheximide (Sigma) (1 h, 30 AM). Cells were then treated with the maximum effective dose of peroxynitrite (100 AM).
Methods and materials Quantitative real-time PCR analysis Cell culture Primary cultures of bovine microvascular endothelial cells (passage 7 –9) were used in these experiments [18,19]. The ONOO treatment was done on serum-starved cultures as described previously [20], with modifications. Briefly, cultures were rinsed in Hanks’ buffered salt solution (HBSS), pH 7.4. HBSS (1.95 ml) was added to each plate and 50 Al concentrated ONOO (Upstate Biotechnology, Lake Placid, NY, USA) diluted to the appropriate concentration in 0.1 N NaOH was rapidly added to the plates while mixing. Cells were incubated in the ONOO -treated buffer for 2 min at 37-C, washed with serum-free medium, and incubated at 37-C for the times indicated. The same volumes of 0.1 N NaOH or decomposed ONOO were used as controls. These control treatments did not alter any of the parameters measured. ONOO concentration was determined by spectrophotometer as described by Zou et al. [21]. In order to evaluate the relative intracellular levels of ONOO reached under these treatment conditions, formation of nitrotyrosine was determined in the ONOO -treated cultures using slot-blot techniques as described previously [20,22]. This analysis showed that the levels of nitrotyrosine formed in cultures treated with 100 –1000 AM ONOO were equivalent to those seen with 2.2 –4.8 mg/ml nitrated BSA. The relative amount of nitrotyrosine formation induced by treatment with 100 AM ONOO was roughly comparable to the levels seen in our previous studies of endothelial cells treated with high glucose or hyperoxia, which were equivalent to 1.8 and 1.6 mg/ml nitrated BSA, respectively [20,22]. To rule out possible ONOO -induced cytotoxic effects, three experiments were conducted using confluent cultures treated with varying doses of ONOO for 6, 12, and 24 h. Cell viability was determined using the Live/Dead Viability/ Cytotoxicity Assay Kit (Molecular Probes, Inc., Eugene,
VEGF mRNA expression was analyzed by quantitative RT-PCR, which was done by using the Cepheid Smart Cycler (Sunnyvale, CA, USA) with a protocol optimized for our primers as previously described [23]. Primers were designed (with the online MIT resource Primer 3 technology) to generate a PCR product of 138 bp and to include a target sequence within exons 3 and 4 of the bovine VEGF gene (Accession No. M32976; Left, 5V-ATTTTCAAGCCGTCCTGTGT-3V, and Right, 5V-TATGTGCTGGCTTTGGTGAG-3V). This sequence recognizes all isoforms of VEGFA and prevents the amplification of multiple products of differing lengths. The housekeeping gene bovine acidic ribosomal protein 1 (ARP-1) (Accession No. AF013214; Left, 5V-TACACCTTCCCACTTGCTGA-3V, and Right, 5VCTCCGACTCCTCCTTTGCTT-3V) was used as an internal standard. Nuclear fractionation To analyze the nuclear translocation of activated STAT3 and Hif-1a, nuclei were isolated from total cell lysates on a 350 mM sucrose gradient as previously described [18,19]. From the nuclear extracts, 25 Ag of protein from each sample was subjected to SDS – PAGE and immunoblotted using anti-STAT3 and anti-Hif-1a antibodies. Immunoblotting analysis Proteins were isolated and quantified as previously described [18]. VEGF was isolated by diluting 100 Ag protein to a volume of 1 ml using 10 mM Tris (pH 7.4) and 100 mM NaCl and incubating with 50 Al of equilibrated heparin –agarose beads (Sigma) as described by Ferrara and Henzel [3] and Hossain et al. [24]. Fifty micrograms of proteins or heparin –agarose-isolated VEGF was electro-
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phoresed on 10% SDS – polyacrylamide or 4– 20% Tris – HCl gradient gels (Bio-Rad Laboratories, Hercules, CA, USA) and then transferred to nitrocellulose membranes and detected with anti-human VEGF165 (Novus Biologicals, Littleton, CO, USA), anti-STAT3, anti-phospho-STAT3 (Cell Signaling Technology, Beverly, CA, USA), or antiHif-1a antibodies (BD-Transduction Labs, San Diego, CA, USA) followed by ECL chemiluminescence (Amersham Biosciences, Piscataway, NJ, USA). ELISA Confluent cultures were incubated in the presence or absence of 100 AM peroxynitrite, degraded peroxynitrite, or control medium for 12 h. Proteins were extracted from and quantified according to our established protocols [18]. Protein samples (50 Ag) were added to wells of a 96-well plate coated with VEGF antibody, incubated at 4-C overnight, and processed according to the manufacturer’s instructions (RayBiotech, Inc., Atlanta, GA, USA). Absorbance was measured in a plate reader at 450 nm. VEGF levels were calculated from absorbance values by comparison with a standard curve prepared by a six-step serial dilution of recombinant VEGF ranging from 8 to 6000 pg/ml. This experiment was repeated twice.
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quantified by measuring the distance between the wound edges before and after injury at five distinct positions (every 5 mm) using computer-assisted microscopy. Adenoviral vectors Replication-deficient adenoviruses expressing a STAT3 DNA binding domain mutant, under the control of the cytomegalovirus (CMV) promoter, were generated using the pAdTrack-CMV vector and AdEasy System [26]. The STAT3 mutant plasmid, pEF-HAStat3D, was a generous gift from Drs. Hirano and Ishihara, Osaka University Medical School, Japan [27]. Competent Escherichia coli were transformed with the STAT3D mutant and the plasmid DNA was recovered by miniprep using the Qiagen QIAprep Spin Miniprep Kit. Plasmid DNA was cut with SalI and NotI restriction endonucleases (New England Biolabs, Inc., Beverly, MA, USA) and the 2500-bp STAT3D was subcloned into the pAdTrack-CMV vector. The pAdTrack vector was electroporated into competent E. coli containing the pAdEasy vector for homologous recombination. After amplification and recovery of the viral vector, viruses were amplified in HEK293 cells, purified using a CsCl gradient, and titered by OD. Statistical analysis
Immunofluorescence Cultures were fixed with 4% paraformaldehyde and then reacted with anti-VEGF antibody followed by Oregon greenlabeled secondary antibody (Molecular Probes). Data were analyzed using the MetaMorph morphometric program (Universal Imaging Corp., West Chester, PA, USA) and fluorescence microscopy to quantify intensity of immunostaining. Specificity of the immunoreaction for VEGF was verified by control studies showing the absence of immunolabeling when the primary antibody was omitted or preadsorbed with the immunizing VEGF peptide [N-terminal sequence (aa 1 –20) of human VEGF]. Cell migration assay To evaluate the potential effects of peroxynitrite-induced increases in VEGF expression, a scratch-wound assay was used to determine the effects of peroxynitrite treatment on endothelial cell migration as described by Dimmeler et al. [25]. Briefly, ‘‘scratch’’ wounds were created by scraping cell monolayers with a precut rubber policeman to produce a wound 2 mm wide and then the wounded cultures were treated with exogenous VEGF (30 ng/ml) or concentrated conditioned medium (0.2 ml) prepared from endothelial cell cultures in 100-mm dishes which had been treated 24 h earlier with either ONOO or decomposed ONOO . The wounded cultures were photographed at specific locations immediately after wounding and 24 h later. Endothelial cell migration from the edge of the injured monolayer was
The results are expressed as the means T SE. Differences between experimental groups were evaluated by ANOVA, and the significance of the differences was determined using the Tukey test for pair-wise comparisons. Significance was defined as p < 0.05.
Results Peroxynitrite stimulation of VEGF expression To determine the effects of ONOO on VEGF expression, serum-starved endothelial cells were treated with varying doses of ONOO for different times and VEGF mRNA was quantified by real-time PCR. The ONOO treatment induced a dose-dependent increase in VEGF mRNA (Fig. 1A), beginning with 50 AM ONOO and reaching a maximum with 100 AM ONOO . This effect was also time-dependent, reaching a maximum within 1 h after treatment and declining to basal levels within 8 h (Fig. 1B). In order to test whether the ONOO -induced increase in VEGF mRNA formation resulted in an increase in VEGF protein, endothelial cells were treated with peroxynitrite (100 AM) and VEGF protein content was analyzed using immunofluorescence, Western blotting, and ELISA techniques. Densitometric analysis of the immunofluorescence and immunoblotting results showed a significant increase in VEGF protein expression in the ONOO -treated cells vs control (Fig. 2). Treatment with degraded ONOO had no
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growth factors. STAT3 is an important regulator of VEGF in angiogenesis [28,29]. Recently, we showed that VEGF autocrine expression in microvascular endothelial cells is regulated by STAT3 [23]. It has also been reported that ROS and drugs that induce oxidative stress can activate STAT3 [30]. To determine if STAT3 is activated by ONOO , we treated endothelial cells with ONOO and determined the effect on phosphorylation of STAT3 at tyrosine 705, which is required for STAT3 activation [31]. These experiments showed that ONOO (100 AM) induced a significant increase in STAT3 tyrosine phosphorylation within 5 min (Fig. 4A). Analyses with increasing concentrations of ONOO also showed a dose-dependent increase in STAT3 tyrosine phosphorylation (data not shown). When STAT proteins are activated by tyrosine phosphorylation they dimerize via their SH2 domains and are shuttled to the nucleus for transcriptional activation of target sequences [32]. To test the effects of ONOO on nuclear translocation of STAT3, we isolated nuclear proteins from treated cells and determined nuclear levels of STAT3 by Western blotting. These experiments showed that ONOO induced increased nuclear levels of STAT3 within 5 min after stimulation (Fig. 4B). Fig. 1. Effects of peroxynitrite on VEGF mRNA. Cells were (A) treated with 0 – 500 AM ONOO and allowed to incubate for 1 h after treatment or (B) treated with 100 AM ONOO and incubated 0 – 8 h after treatment, and effects on VEGF mRNA levels were determined by quantitative real-time PCR. The results are expressed as the ratio VEGF mRNA to ARP-1 mRNA T SE for three separate experiments. *p < 0.05 compared with control values.
effect on VEGF protein levels in either assay. Quantitation of VEGF protein levels using ELISA confirmed a significant (3.8-fold, p < 0.001) increase in VEGF protein formation in the ONOO -treated cells. The VEGF level in cells treated with ONOO (100 mM, 12 h) was 96 T 11 pg/ ml compared with 25 T 9 pg/ml in the untreated control cells and 17 T 5 pg/ml in the cells treated with decomposed ONOO . In order to evaluate the potential biological effects of the peroxynitrite-induced increases in VEGF protein expression, experiments were performed to determine the functional effects of ONOO treatment on endothelial cell migration. The results of experiments using a scratch-wound cell migration assay showed that medium conditioned by ONOO -treated cultures caused a significant increase in cell migration compared with medium from control cultures treated with decomposed ONOO (Fig. 3). This effect of ONOO was roughly comparable with that of exogenous VEGF. Activation of STAT3 by peroxynitrite STAT proteins are a class of latent cytoplasmic transcription factors that regulate the expression of genes involved in cellular growth induced by cytokines and
Fig. 2. Effects of ONOO on VEGF protein expression. Microvascular endothelial cells were treated with or without ONOO or degraded ONOO (100 AM) and incubated for 12 h in serum-free medium and effects on levels of VEGF protein were determined by (A) immunocytochemistry or (B) Western blotting. Results are expressed as relative optical density means T SE. n = 5 in A, n = 3 in B, *p < 0.05 compared with control values.
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Fig. 3. Effects of ONOO on cell migration. Cultures of microvascular endothelial cells were wounded as described under Methods and materials and treated with VEGF (30 ng/ml) or medium conditioned by cultures treated with or without ONOO (PN) or degraded ONOO (dPN) (100 AM). (A) Cultures were photographed immediately after wounding and 24 h later. (B) Migration was quantified by measuring the distance between the wound edges before and after treatment. The results are expressed as the % of wound closure T SE for three separate experiments. *p < 0.05 compared with control values.
Effects of peroxynitrite on hypoxia-inducible factor-1 (HIF-1) activation We next examined the potential role of HIF-1 in ONOO induced VEGF transcription. HIF-1 is a transcription factor that regulates a number of genes under conditions of low oxygen tension, including VEGF. To be activated, Hif-1a, the inducible subunit of HIF-1, must undergo cytosolic stabilization and accumulation, which is followed by nuclear translocation. It is generally thought that this stabilization does not occur in the absence of hypoxia and that Hif-1a is degraded. However, recent studies have shown that formation of reactive oxygen and nitrogen species plays a role in the activation of Hif-1a [33]. Therefore, to determine the effects of ONOO on the regulation of Hif-1a, endothelial cells were treated with 100 AM ONOO for 0, 5, 15, 30, and 60 min. Hif-1a protein was not detected in the total cell lysates (Fig. 5A), indicating that ONOO did not stabilize or
increase cytosolic levels of Hif-1a protein. To activate transcription, HIF-1 must be translocated to the nucleus and bind the hypoxia response element in the promoter of its target genes [34]. To determine if ONOO induces nuclear translocation of HIF-1, nuclear proteins isolated from treated cells and levels of Hif-1a within the nucleus were analyzed by immunoblotting. As seen in Fig. 5B, Hif-1a was not detected in the nucleus of the ONOO -treated cells. Requirement of STAT3 activity for peroxynitrite-mediated VEGF expression The above results suggest that STAT3 and not HIF-1 is involved in ONOO -mediated activation of VEGF expression. To test whether STAT3 function is required for the ONOO effect, a STAT3 DNA binding domain mutant from pEF-HAStat3D was subcloned into the pAdTrack-CMV vector in order to produce a dominant-negative adenovirus
Fig. 4. Effects of ONOO on activation and nuclear translocation of STAT3. (A) Microvascular endothelial cells were treated with ONOO (100 AM) and STAT3 activation was determined after 0 – 60 min by Western blotting of total cell lysates using an antibody against phosphotyrosine (705) STAT3. Blots were reprobed with STAT3 antibody to demonstrate equal loading. (B) Nuclear translocation of STAT3 was determined by Western blotting of isolated nuclear proteins using an antibody against STAT3. Results were quantified by densitometry and results are expressed as means T SE for three separate experiments. *p < 0.05 compared with untreated controls.
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The ONOO effect was significantly blunted in cells transduced with AdSTAT3D. The levels of VEGF mRNA in cells transduced with AdSTAT3D but not treated with ONOO were no different from that in the untreated AdGFP-transduced control cells. Effects of inhibitors of transcription on peroxynitrite-mediated VEGF expression Fig. 5. Effects of ONOO on HIF-1 activation and nuclear translocation. (A) Microvascular endothelial cells were incubated with ONOO (100 AM) and HIF-1 activation was assayed after 0 – 60 min by Western analysis of Hif-1a protein levels in total cell lysates. (B) Nuclear translocation of HIF-1 was determined by Western analysis of isolated nuclear proteins with Hif1a antibody. As a positive control (C+) for HIF-1 activation, cells were treated with cobalt chloride (150 AM) for 4 h. All blots are representative of three separate experiments.
(AdSTAT3D). The pAdTrack-CMV vector drives expression of green florescent protein (GFP), which allows tracking of cell transduction levels. Endothelial cells were transduced with the adenovirus expressing GFP only or AdSTAT3D at an estimated multiplicity of infection (m.o.i.) of 30 viral particles per cell. GFP expression was tracked for 12 h at which time an estimated 95% or more of the cells were expressing GFP (data not shown). Overexpression of dominant-negative STAT3 was verified by immunoblotting analysis using anti-HA and anti-STAT3 antibodies (data not shown). The cells were switched to serum-free medium for 12 h and then treated with or without ONOO (100 AM, 1 h) and the effects on VEGF mRNA were determined by quantitative real-time PCR. These experiments showed a significant increase in VEGF mRNA expression after ONOO treatment of cells expressing only GFP (Fig. 6).
Fig. 6. Effects of dominant-negative STAT3 on ONOO -induced VEGF expression. Microvascular endothelial cells were transduced with adenoviruses expressing STAT3 with a DNA binding domain mutation at an m.o.i. of 30. Cells were transduced with adenovirus expressing only GFP or GFP and STAT3D (DNA binding domain mutant) and treated with or without ONOO (100 AM) and incubated in serum-free medium for 1 h. VEGF mRNA levels were determined by quantitative real-time PCR. Results are expressed as the ratio of VEGF mRNA to ARP-1 mRNA T SE for three separate experiments. *p < 0.002 compared with control values. #p < 0.05 compared with ONOO -treated AdGFP.
VEGF expression is a complex process that requires transcriptional and posttranscriptional regulation [35,36]. In order to evaluate whether ONOO -induced increases in activation of STAT3 and induction of VEGF mRNA formation require increased transcription, endothelial cells were treated with the maximum effective dose of peroxynitrite (100 AM) in the presence or absence of two different inhibitors of transcription, actinomycin D and 5,6-dichlorobenzimidazole riboside (DRB). Actinomycin D specifically inhibits RNA polymerase through complex formation with deoxyguanosine residues in DNA primers. DRB inhibits transcription by blocking the activity of casein kinase II, which is required for activity of RNA polymerase II. The ONOO -induced increases in VEGF mRNA were completely inhibited in the cells treated with either actinomycin D or DRB (Fig. 7). This suggests that ONOO induced increases in VEGF required mRNA transcription. By contrast, treatment of the cultures with the protein synthesis inhibitor cycloheximide had no significant effect on the action of ONOO in increasing VEGF mRNA, indicating that the increase in VEGF mRNA does not require synthesis of new proteins.
Fig. 7. Effects of inhibitors of transcription and protein synthesis on ONOO induced VEGF mRNA. Microvascular endothelial cells were treated with or without ONOO (PN, 100 AM) and incubated for 1 h in the presence or absence of transcriptional inhibitors actinomycin D (Act-D, 4 Ag/ml) and DRB (50 Ag/ml) and the protein synthesis inhibitor cycloheximide (CHX, 30 AM). Cells were preincubated with the transcriptional inhibitors for 3 h before peroxynitrite treatment and with the protein synthesis inhibitor for 1 h. Realtime, quantitative PCR was used to measure levels of VEGF mRNA. Results are expressed as the ratio of VEGF mRNA to ARP-1 mRNA T SE for four separate experiments for actinomycin D and three separate experiments for DRB and CHX. *p < 0.001 compared with control (lane 1) values and #p < 0.001 compared with peroxynitrite (PN) alone.
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Discussion Increased expression of VEGF has been implicated in a variety of disease conditions characterized by pathological vascular growth, including diabetic microvascular disease, atherosclerosis, and tumor angiogenesis. Overexpression of VEGF has been correlated with increased levels of oxidative stress and with formation of the ONOO biomarker nitrotyrosine [2,4,9,14 –16]. In this study we tested the hypothesis that ONOO has a direct effect in stimulating the expression of VEGF in vascular endothelial cells. Here we show that treatment of primary vascular endothelial cells with exogenous ONOO induces a dose- and time-dependent increase in VEGF mRNA which is followed by increases in VEGF protein formation. Moreover, medium conditioned by cells treated with ONOO causes increases in endothelial cell migration, indicating that the peroxynitrite effect is functionally relevant. These data, together with previous work showing that ONOO is up-regulated in diabetes and that treatments which reduce ONOO formation prevent VEGF overexpression [2], strongly support the hypothesis that ONOO directly stimulates VEGF expression. The maximum increase in VEGF expression occurred with ONOO treatment at a concentration of 100 AM. Although this concentration exceeds that which is likely to occur intracellularly under either physiological or pathophysiological conditions, this is not surprising because the half-life of ONOO at pH 7.4 is on the order of seconds [37]. Both exposure time and concentration of exogenous ONOO are critical determinants for mimicking the effects of endogenous ONOO . Thus, much higher concentrations of exogenous ONOO may be needed to achieve biological responses similar to those seen with endogenous ONOO , which is continuously produced at low concentrations. To understand the molecular mechanisms of ONOO -induced VEGF expression we analyzed the activation of two regulators of VEGF expression: the transcription factors STAT3 and HIF-1. Our data demonstrated that ONOO induces the activation of STAT3 and that STAT3 activation is required for the effects of ONOO in stimulating VEGF expression. The STAT protein family of latent cytoplasmic transcription factors was originally thought to provide selective signaling because each member was activated by a different cytokine receptor. However, STAT3 is now known to be activated by a number of cytokines, growth factors, and oncogenes and to participate in different signal pathways in different cell types [38]. We have recently shown that STAT3 activation plays an important role in autocrine VEGF expression in microvascular endothelial cells [23]. Others have shown that STAT3 is activated in a wide variety of cancers and that its activation increases both VEGF expression and tumor angiogenesis [39]. Our present findings suggest that STAT3 also has a key role in ONOO -mediated induction of VEGF expression in microvascular endothelial cells. Unpublished
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studies using primary cultures of aortic smooth muscle cells indicate that ONOO treatment induces similar patterns of STAT3 activation and VEGF mRNA increases, suggesting that these effects of ONOO also apply to other vascular cell types (D.H. Platt, M. Bartoli, and R. Caldwell, unpublished results). Recent studies have shown that oxidative stress can activate STAT3 by phosphorylation of tyrosine 705 within the activation domain. Furthermore, studies of the effects of oxidative stress on tyrosine kinase activity show that receptor and nonreceptor tyrosine kinases, which can directly interact with and activate STAT3, can also be activated by ONOO [40]. Here we have shown that ONOO causes STAT3 activation as indicated by its tyrosine phosphorylation and nuclear translocation and that overexpression of dominant-negative STAT3 inhibits ONOO induced VEGF expression. These data suggest that ONOO mediated activation of STAT3 could have a key role in triggering the up-regulation of VEGF expression in diabetic microangiopathy. Studies are now in progress using animal and tissue culture models of diabetes to test this hypothesis. Our experiments showed that the ONOO treatment had no effect on either the cytosolic stability or the nuclear translocation of HIF-1, indicating that activation of HIF-1 probably does not play a role in the ONOO -induced increase in expression of VEGF. Low oxygen tension is known to stimulate cells to express VEGF by inducing activation of HIF-1. However, recent studies indicate that reactive oxygen or reactive nitrogen species can also activate HIF-1 [33,41]. These previous studies found that, S S whereas either O2 or NO alone could induce stabilization of Hif-1a, their contemporaneous production, which could favor ONOO formation, failed to induce HIF-1 activation. This is consistent with our present results. Studies have shown that hypoxia-induced increases in VEGF expression are due in large part to increases in the stability of VEGF mRNA [42]. Hydrogen peroxide- or superoxide-induced increases in VEGF mRNA levels in retinal pigment epithelial cells have been shown to be mediated exclusively by increases in mRNA stability [43]. Further investigation will be needed to determine whether ONOO increases VEGF mRNA by increasing transcriptional activity and/or by enhancing mRNA stability. However, our finding that inhibitors of transcription totally blocked the ONOO effect, whereas an inhibitor of protein synthesis was without effect, implies that transcriptional activation is likely to be involved in the process. In conclusion, ONOO has been implicated in vascular dysfunction in cardiovascular disease and the ONOO biomarker nitrotyrosine has been observed in both diabetes and atherosclerosis. Overexpression of VEGF and increased oxidative stress also occur under both conditions and are correlated with disease progression. Previous studies in models of tumor angiogenesis have shown that ONOO formation is correlated with increases in VEGF expression and that treatment of a mast cell tissue culture line with
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exogenous ONOO is associated with increases in VEGF mRNA [14,17]. Our study is the first to show that ONOO can directly stimulate VEGF expression in microvascular endothelial cells. We also show that ONOO -induced VEGF expression is mediated by activation of the transcription factor STAT3, suggesting a specific molecular role for STAT3 in VEGF overexpression and disease progression in vascular diseases characterized by increased oxidative stress.
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Acknowledgments This work was supported in part by NIH Grants R01NEI-04618 and R01-NEI-11766 and American Heart Association Grant 0365181B.
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