www.elsevier.com/locate/freeradbiomed

Free Radical Biology & M

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,*

aVascular Biology Center, Medical College of Georgia, Augusta, GA 30912, USAbDepartment of Pathology, Medical College of Georgia, Augusta, GA 30912, USA

cDepartment of Pharmacology and Toxicology, Medical College of Georgia, Augusta, GA 30912, USAdDepartment of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, GA 30912, USA

eDepartment of Ophthalmology, Medical College of Georgia, Augusta, GA 30912, USA

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 mRNAwas 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

by the diffusion-limited reaction of O2S� and nitric oxide

(SNO), 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

0891-5849/$ - see front matter D 2005 Elsevier Inc. All rights reserved.

doi:10.1016/j.freeradbiomed.2005.06.015

Abbreviations: VEGF, vascular endothelial growth factor; ROS,

reactive oxygen species; ONOO�, peroxynitrite;SNO, nitric oxide; O2

S�,

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: rcaldwel@mail.mcg.edu (R.B. Caldwell).

inflammatory responses, including induction of the angio-

genic 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

edicine 39 (2005) 1353 – 1361

D.H. Platt et al. / Free Radical Biology & Medicine 39 (2005) 1353–13611354

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.

Methods and materials

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 Alconcentrated 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 spec-

trophotometer as described by Zou et al. [21].

In order to evaluate the relative intracellular levels of

ONOO� reached under these treatment conditions, forma-

tion 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,

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-dichlorobenzimi-

dazole 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).

Quantitative real-time PCR analysis

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 techno-

logy) 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-ATTTTCAAGCC-GTCCTGTGT-3V, and Right, 5V-TATGTGCTGGCTTTG-GTGAG-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, 5V-CTCCGACTCCTCCTTTGCTT-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 Agprotein 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-

D.H. Platt et al. / Free Radical Biology & Medicine 39 (2005) 1353–1361 1355

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 anti-

Hif-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 over-

night, and processed according to the manufacturer’s

instructions (RayBiotech, Inc., Atlanta, GA, USA). Absorb-

ance 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.

Immunofluorescence

Cultures were fixed with 4% paraformaldehyde and then

reacted with anti-VEGF antibody followed by Oregon green-

labeled 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 immunos-

taining. Specificity of the immunoreaction for VEGF was

verified by control studies showing the absence of immuno-

labeling when the primary antibody was omitted or pre-

adsorbed 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

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

DNAwas 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

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 expres-

sion, 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 techni-

ques. 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

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) immunocy-

tochemistry 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.

Fig. 1. Effects of peroxynitrite on VEGFmRNA. 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 VEGFmRNA to ARP-1mRNA TSE for three separate experiments. *p < 0.05 compared with control values.

D.H. Platt et al. / Free Radical Biology & Medicine 39 (2005) 1353–13611356

effect on VEGF protein levels in either assay. Quantitation

of VEGF protein levels using ELISA confirmed a signifi-

cant (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 expres-

sion, experiments were performed to determine the func-

tional 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 tran-

scription factors that regulate the expression of genes

involved in cellular growth induced by cytokines and

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 transcrip-

tional 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. 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.

D.H. Platt et al. / Free Radical Biology & Medicine 39 (2005) 1353–1361 1357

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 for-

mation 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

Fig. 4. Effects of ONOO� on activation and nuclear translocation of STAT3. (A)

STAT3 activation was determined after 0–60 min by Western blotting of total cell

reprobed with STAT3 antibody to demonstrate equal loading. (B) Nuclear transl

proteins using an antibody against STAT3. Results were quantified by densitomet

*p < 0.05 compared with untreated controls.

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 expres-

sion. 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

Microvascular endothelial cells were treated with ONOO� (100 AM) and

lysates using an antibody against phosphotyrosine (705) STAT3. Blots were

ocation of STAT3 was determined by Western blotting of isolated nuclear

ry and results are expressed as means T SE for three separate experiments.

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 Hif-

1a 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.

D.H. Platt et al. / Free Radical Biology & Medicine 39 (2005) 1353–13611358

(AdSTAT3D). The pAdTrack-CMV vector drives expres-

sion 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 adenovi-

ruses 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.

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

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 peroxyni-

trite (100 AM) in the presence or absence of two different

inhibitors of transcription, actinomycin D and 5,6-dichlor-

obenzimidazole riboside (DRB). Actinomycin D specifi-

cally 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 actino-

mycin 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 onONOO�-

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 preincubatedwith the transcriptional inhibitors for 3 h before

peroxynitrite treatment and with the protein synthesis inhibitor for 1 h. Real-

time, quantitative PCR was used to measure levels of VEGFmRNA. 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.

D.H. Platt et al. / Free Radical Biology & Medicine 39 (2005) 1353–1361 1359

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 over-

expression [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 patho-

physiological 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 acti-

vation of two regulators of VEGF expression: the tran-

scription 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 micro-

vascular 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

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 over-

expression 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,

whereas either O2S�or

SNO 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 transcrip-

tional activity and/or by enhancing mRNA stability. How-

ever, 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

D.H. Platt et al. / Free Radical Biology & Medicine 39 (2005) 1353–13611360

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 tran-

scription factor STAT3, suggesting a specific molecular role

for STAT3 in VEGF overexpression and disease progression

in vascular diseases characterized by increased oxidative

stress.

Acknowledgments

This work was supported in part by NIH Grants R01-

NEI-04618 and R01-NEI-11766 and American Heart

Association Grant 0365181B.

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