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* Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, Athens, Georgia, USA;
Department of Pharmacology and Toxicology,
Vascular Biology Center,
Department of Oral Biology and Maxillofacial Pathology,
|| Department of Ophthalmology, Medical College of Georgia, August, Georgia, USA;
¶ Department of Ophthalmology, University of South Carolina, Columbia, South Carolina, USA; and
# VA Medical Center, Augusta, Georgia, USA
1Correspondence: Program in Clinical and Experimental Therapeutics, College of Pharmacy, University of Georgia, CJ-1033, 1120 15th Street, Augusta, GA 30912, USA. E-mail: aelremessy{at}mcg.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: angiogenesis • Src • redox signaling
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3)
. The discovery that ROS function as physiological regulators of tyrosine kinase receptor signaling cascades has highlighted the possible mechanism underlying the growth-regulating and tumor-promoting activities of ROS (4)
. In addition, it has become evident that intracellular ROS are necessary for optimal propagation of VEGF-mediated mitogenic and angiogenic signals. Binding to VEGFR2 initiates its tyrosine phosphorylation and results in activation of key signaling cascades involved in cell survival, proliferation, migration, vasorelaxation, and vascular permeability (5
6
7)
.
Indeed, an emerging concept in research on VEGF’s angiogenic signaling in vascular endothelial cells is that superoxide anion and hydrogen peroxide may serve as intracellular messengers involved in autophosphorylation of VEGFR2 (8
, 9)
. Yet, in these studies the well-known role of nitric oxide (NO) in mediating VEGF’s angiogenic function (10
11
12)
in vitro and in vivo and its possible interaction with superoxide anion were not explored. Of note, the rate constant for the reaction of superoxide anion with NO is 10-fold faster than with SOD, leading to the diffusion-controlled formation of peroxynitrite (13)
. Peroxynitrite is a powerful oxidant that causes protein modification via oxidation of protein-associated thiol groups or nitration of tyrosine residues.
So far, the focus of research on peroxynitrite during vascular disease has emphasized its potential in inhibiting normal cell signaling pathways. Our studies showed that inhibiting peroxynitrite formation and tyrosine nitration blocks increases of VEGF expression, vascular permeability, and restored VEGF survival signal in models of experimental diabetes and hyperglycemia (14
15
16
17)
. Other studies indicate that low, controlled levels of peroxynitrite are generated in nonpathological conditions to trigger specific cellular pathways (18
19
20
21)
. Recently, we have shown that relatively low levels of peroxynitrite up-regulate VEGF mRNA and protein expression in endothelial cells (20)
. These findings suggest a possible loop effect of VEGF signaling and peroxynitrite formation, which prompted us to investigate the role of intracellular peroxynitrite in mediating VEGF’s signaling linked to angiogenic-related responses such as cell migration, proliferation, and tube morphogenesis and to delineate the molecular mechanisms by which peroxynitrite transduces the signal in endothelial cells.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The cells (passages 6–8) were plated on gelatin-coated dishes, grown to confluence, then treated. Human endothelial cells (HUVEC) were obtained from VEC Technologies (Rensselaer, NY, USA) and cultured according the manufacturer’s protocol. Experiments were performed using cells between passages 2 and 3. Cell culture reagents were purchased from Gibco BRL (Grand Island, NY, USA) except for fetal bovine serum (FBS), which was purchased from HyClone Laboratories, Inc. (Logan, UT, USA), and CS-C Complete Medium, from Cell Systems Corporations (Kirkland WA, USA). Inhibitors, including L-NAME, uric acid, superoxide dismutase-polyethylene glycol (SOD-PEG), catalase-polyethylene glycol (CAT-PEG), epicatechin, and N-acetyl cysteine (NAC), were obtained from Sigma-Aldrich (St. Louis, MO, USA). The specific peroxynitrite decomposition catlayst [III] porphyrin 5,10,15,20-tetrakis[4-sulfonatopheny]prophyrinato iron chloride (FeTPPs) was obtained from Calbiochem (La Jolla, CA, USA).
Peroxynitrite treatment
Cells were switched to serum-free medium, treated with 1 µM peroxynitrite, and cultured for the times indicated. Peroxynitrite was purchased from Upstate Biotechnology (Lake Placid, NY, USA). Stock concentrations of peroxynitrite were freshly prepared in 0.1N NaOH. Peroxynitrite concentration was determined by spectrophotometer as described by Zou et al. (18)
. An equal amount of 0.1N NaOH or decomposed peroxynitrite was used for control experiments. Neither affected any of the parameters analyzed.
Determination of NO, superoxide anion, and peroxynitrite in microvascular endothelial cells
VEGF-induced NO and superoxide anion formation was assessed using confocal microscopy to detect the oxidation of diaminofluorescin (DAF) or dihydroethidine (DHE) (Molecular Probes, Eugene, OR, USA), respectively. Cells were seeded at a density of 1 x 105/ml. After attachment, medium was switched to serum-free medium overnight. The cells were incubated with either DAF or DHE for 10 min and the baseline was established in untreated cells (excitation at 485 nm and emission at 535 nm), followed by subsequent stimulation with VEGF (30 ng/ml). Dose-response studies showed that 30 ng/ml was the optimum dose of VEGF to stimulate superoxide anion in microvascular endothelial cells and that dose was used throughout the entire study. Fluorescence intensity of the images was analyzed using an imaging system (MetaMorph; Universal Imaging Corp., West Chester, PA, USA) and the oxidation level was calculated based on relative fluorescence per cell number. For peroxynitrite formation, confocal microscopy was used to detect the oxidation of 2,7-dihydrodichloroflurescin diacetate (DHCF) (Molecular Probes, Eugene, OR, USA). DCF is the oxidation product of DHCF and is widely used as a general marker of cellular oxidation by hydroxyl radicals, hydrogen peroxide, and peroxynitrite. The cultures were treated with DHCF (5 µM) for 1 h, then exposed to VEGF (30 ng/ml) in the presence or absence of various inhibitors. Confocal microscopy was used to follow DHCF oxidation (excitation at 485 nm and emission at 535 nm). Fluorescence intensity of the images was analyzed using an imaging system (MetaMorph; Universal Imaging Corp., West Chester, PA, USA) and the oxidation level was calculated based on relative fluorescence per cell number. Further experiments were carried out to establish a standard curve of DCF fluorescence using serial dilution of peroxynitrite (0.1 µM to 1 mM). The results showed that VEGF (30 ng/ml) stimulates peroxynitrite formation in the range of 1–10 µM in microvascular endothelial cells. For control studies, the peroxynitrite scavenger uric acid was used to demonstrate that DCF fluorescence reflects peroxynitrite formation.
Western blot analysis
Microvascular or human endothelial cells were harvested after various treatments and lysed in modified RIPA buffer (20 mM Tris, 2.5 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 40 mM NaF, 10 mM Na4P2O7, and 1 mM PMSF) for 30 min on ice. Insoluble material was removed by centrifugation at 12,000 g at 4°C for 30 min. Antibodies for phospho-VEGFR2, VEGFR2, phospho-Src, and Src were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). For Western blot analysis, 50 µg of total protein was boiled in 2 x Lammeli sample buffer, separated on a 10–12% SDS-polyacrylamide gel by electrophoresis, transferred to nitrocellulose, and reacted with specific antibody. The primary antibody was detected using a horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit antibody (Amersham Biosciences, Buckinghamshire, UK) and enhanced chemiluminescence. Intensity of immunoreactivity was measured using densitometry software Image J (from NIH website).
Tube formation assay
We used a 3-dimensional tubulogenesis assay to study the effects of various inhibitors on VEGF-induced tube formation. Microvascular or human endothelial cells (106) were suspended in 1 ml of a 2.5 mg/ml solution of neutralized rat tail collagen type I (BD Biosciences, Bedford, MA, USA) containing 1% FBS. Aliquots (30 µl) of the cell suspension were placed onto Nitex assay discs (0.22" O.D., Tetko Inc., NY, USA) and allowed to polymerize for 90 min at 37°C. Discs were then placed in 96-well plates prefilled with EGM-2 medium bullet kit (Cambrex, Baltimore, MD, USA) containing 1% FBS, 20 ng/ml phorbol myristate acetate (Sigma) in the presence or absence of VEGF (30 ng/ml). Various inhibitors were then added to control or VEGF-stimulated cultures. Medium was replaced daily for 5 days. Discs were fixed in 4% paraformaldehyde (Sigma), stained briefly (15–30 s) with 0.5% crystal violet (Sigma), destained with distilled water for 2–5 min, and photographed under 200 x magnification using confocal microscopy (Carl Zeiss Laser Scanning Microscope LSM 510 Meta Version 3.2). Tube length measurements were determined using an imaging system (MetaMorph; Universal Imaging Corp., West Chester, PA, USA).
Cell migration assay
Microvascular endothelial cells were allowed to reach confluence in growth medium, then switched to serum-free medium prior to addition of the test agents. The monolayer was wounded with a single sterile cell scraper of constant diameter. Cells were switched to serum-free medium and treated with VEGF in the presence or absence of various inhibitors. Images of wounded areas were taken immediately after the treatment and after 24 or 48 h. Cells crossing the wound were counted per unit time.
Cell proliferation assay
Cells were seeded at a density of 0.5 x 105/ml, switched to medium containing 0.5% FBS, and incubated overnight. Cells were incubated with and without VEGF, peroxynitrite, and inhibitors in medium containing 0.2% FBS for 72 h. After trypsinization, the cell number was determined using a hemacytometer.
Chorioallantoic membrane (CAM) assay
Angiogenic assay with chick CAM was performed as described previously (23)
. Fertilized chick embryos were incubated at 37°C with 70% humidity for the entire study. After 48 h, a hole was drilled over the air sac at the end of the eggs and an avascular zone was identified on the CAMs. A 1 x 1 cm window in the shell was made to expose the CAM. The windows were sealed with clear tape. Sterile gel foam plugs (Pharmacia-Upjohn, Kalamazoo, MI, USA) of identical size were impregnated with VEGF (30 ng/ml), peroxynitrite (1 µM), or vehicles transferred onto the CAM at day 9, incubated until day 12, and observed with a Zeiss stereoscope microscope. Stimulation of angiogenesis was estimated by the number of new blood vessels growing toward the gel foam. The CAM was fixed with 4% formaldehyde and mounted on slides, and the vascular intensity was analyzed with a computer-assisted image program (NIH Image J).
Mouse model of hypoxia-induced neovascularization
This is an established model in our lab to study retinal neovascularization (24)
using the previously described protocol of Smith and colleagues (25)
. On postnatal day 7 (P7), C57BL/6 mice were placed in 75% oxygen (hyperoxia) and after 5 days (P12) returned to room air. The hyperoxia exposure causes obliteration of the developing vessels in the posterior retina. This results in relative hypoxia and causes retinal neovascularization that peaks at P17. Age-matched mice raised in room air were used as normoxia controls. All experimental procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Institutional Committee for the Use of Animals in Research. Mice (n=8 in each group) were treated with 50 µl of FeTPPs (1 mg/kg/day, i.p.), epicatechin (10 mg/kg/day i.p.), or NAC (150 mg/kg/day, i.p.) from P12 through P17. A group of mice injected with PBS (50 µl) served as vehicle controls.
Analysis of neovascularization
Vascular distribution was analyzed in the neonatal retinas by using flat-mount preparations labeled with biotinylated Griffonia simplicifolia isolectin B4 (GSI) and Texas red-conjugated avidin D as described previously (24)
. Retinas were viewed with LSM 510 confocal microscopy (Carl Zeiss, Thornwood, NY, USA) and the images were captured in digital format (Spot System; Diagnostic Instruments, Sterling Heights, MI, USA). The areas of retinal neovascularization were quantified from the digital images in masked manner, using IPLab Spectrum Scientific Image System (Signal Analytics, Vienna, VA, USA).
Statistical analysis
Results are expressed as mean ± SE. Differences among experimental groups were evaluated by ANOVA and the significance of differences between these groups was assessed by Fisher’s post hoc least significant difference test. *Significance was defined at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Peroxynitrite mediates VEGF-induced endothelial cell tube formation, migration, and proliferation
To test the hypothesis that peroxynitrite mediates VEGF’s angiogenic function, we determined the effects of inhibiting peroxynitrite on VEGF-stimulated tube formation, migration, and proliferation in cultured endothelial cells. Studies using an in vitro model of capillary morphogenesis in 3-dimensional type I collagen matrix showed that the specific peroxynitrite decomposition catalyst (FeTPPs, 2.5 µM) completely blocked VEGF-induced tube formation (Fig. 1
A). As shown in Fig. 1B, C
, FeTPPs also blocked the action of VEGF (30 ng/ml) in stimulating endothelial cell migration and growth. Similar results were obtained with a combination of SOD-PEG (100 U/ml) and L-NAME (0.5 mM). FeTPPs did not alter cell migration, growth, or alignment into tube-like structures in normal controls.
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Peroxynitrite mimics VEGF’s angiogenic function in vitro and in vivo
To confirm the role of peroxynitrite in mediating VEGF’s angiogenic function, cells were treated with a serial dilution of peroxynitrite from 0.1 µM to 100 µM. As shown in Fig. 2
A, peroxynitrite caused a dose-dependent increase in cell growth. The maximum effect was obtained at 1 µM (2.5-fold, P<0.05) compared with cells treated with decomposed peroxynitrite (DPN). A similar pattern of dose-dependent effects of peroxynitrite in stimulating tube-like structures was obtained using growth factor-reduced Matrigel 3-dimensional cultures (data not shown). Next we tested the effects of 1 µM peroxynitrite in stimulating new blood vessel growth in vivo in a chick CAM assay. As shown in Fig. 2B
, vascular density analysis showed that peroxynitrite (1 µM) mimicked VEGF (30 ng/ml) in stimulating new capillary growth (80% and 60%, respectively) in the treated CAMs compared with controls treated with vehicle. These results indicate that low levels of peroxynitrite can mimic VEGF’s action and suggest a physiological role of peroxynitrite in mediating VEGF’s angiogenic function.
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Peroxynitrite sustains VEGF-stimulated autophosphorylation of VEGFR2
The biological effects of VEGF in endothelial cells are mediated primarily via VEGFR2. On ligand binding, VEGFR2 is rapidly autophosphorylated, creating docking sites for downstream signaling effectors. To evaluate the importance of ligand-induced peroxynitrite formation for VEGFR2 autophosphorylation, cells were pretreated with the specific peroxynitrite decomposition catalyst FeTPPs and subsequently exposed to VEGF. Immunoblotting using antibodies against phospho-VEGFR2 showed that VEGF (30 ng/ml) caused a time-dependent activation of VEGFR2 within 1 min, which was sustained at 5 min and resumed at 30 min (Fig. 3
A). FeTPPs (2.5 µM) reduced but did not eliminate the immediate phosphorylation (1 min), but inhibited the sustained signal (5–30 min), suggesting a positive feedback action of peroxynitrite to sustain VEGF signaling. Controls treated with FeTPPs showed phosphorylation levels similar to those of untreated controls. As shown in Fig. 3A
, exogenous peroxynitrite (1 µM) also caused an immediate increase in phosphorylation of VEGFR2, which was sustained for 30 min. FeTPPs blocked this effect. Vehicle and decomposed peroxynitrite had no significant effects on VEGFR2 phosphorylation (data not shown). The results suggest an autophosphorylation loop in which VEGF activation of VEGFR2 stimulates intracellular peroxynitrite formation, which positively feeds back to sustain the receptor activation and propagate the signal to the downstream targets.
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Hydrogen peroxide does not mediate early phase of VEGFR2 autophosphorylation
To verify the role of NO, hydrogen peroxide, or superoxide in the early phase activation of VEGFR2, endothelial cells were pretreated with the general NOS inhibitor (L-NAME, 0.5 mM), hydrogen peroxide decomposer (catalase, 1000 U/ml), or a combination of L-NAME and the superoxide oxide anion dismutase (SOD, 100 U/ml), followed by VEGF stimulation. As shown in Fig. 3B
, inhibiting NOS alone or in combination with SOD completely blocked the early phase of VEGF-induced autophosphorylation (1–5 min). Inhibiting hydrogen peroxide did not alter VEGF-induced phosphorylation. These results confirmed the involvement of NO and peroxynitrite but not hydrogen peroxide in mediating early activation of VEGFR2.
Nitration inhibitor does not alter VEGFR2 phosphorylation
We next studied the mechanism by which peroxynitrite, a powerful oxidizing and nitrating agent, stimulates tyrosine phosphorylation. Epicatechin, a dietary flavanol, is a cell-permeable nitration inhibitor that selectively blocks peroxynitrite’s action of nitrating tyrosine residues but has no effect on thiol oxidation (26
, 27)
. As shown in Fig. 3B
, selective blockade of tyrosine nitration using epicatechin (100 µM) did not alter the action of VEGF in triggering tyrosine phosphorylation of VEGFR2. These effects were also mimicked by treatment of cells stimulated with 1 µM peroxynitrite (data not shown). These data together with the above finding that blocking peroxynitrite formation inhibited VEGF-induced autophosphorylation suggest that peroxynitrite formation is essential to sustain and propagate VEGF’s signal via a mechanism that does not involve tyrosine nitration.
Blocking tyrosine nitration enhances tyrosine phosphorylation of c-Src
To further evaluate the impact of peroxynitrite-induced nitration on VEGF signaling, we next examined the effects of blocking peroxynitrite or tyrosine nitration on VEGF-dependent activation of c-Src kinase. Our results showed that VEGF induced a time-dependent increase in phosphorylation of c-Src that was completely blocked by treatment with FeTPPs (2.5 µM). In contrast, VEGF-induced c-Src phosphorylation was enhanced by the selective nitration inhibitor epicatechin (100 µM) (Fig. 4
A). Similar results were obtained for peroxynitrite (1 µM) (Fig. 4B
). DPN had no significant effect on c-Src phosphorylation (data not shown). These results confirm the above finding that VEGF-induced peroxynitrite formation propagates the VEGF signal by a mechanism that does not involve tyrosine nitration.
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VEGF’s angiogenic signal in human endothelial cells requires peroxynitrite-mediated oxidation but not nitration
Next, we verified the previous finding using human endothelial cells (HUVEC). As shown in Fig. 5
A, VEGF caused immediate activation of VEGFR2 that was sustained at 5 min and resumed at 30 min (top panel). VEGF-induced autophosphorylation was maintained in cells pretreated with the specific nitration inhibitor epicatechin (100 µM) (top panel). Pretreatment with FeTPPs (2.5 µM) reduced but did not eliminate the immediate phosphorylation (1 min), but blocked the sustained signal (5–30 min) (lower panel). These results confirm our previous finding that VEGF-stimulated intracellular peroxynitrite formation sustains VEGF autophosphorylation and that tyrosine nitration is not required for mediating VEGF-induced autophosphorylation. To test whether peroxynitrite could be mediating the VEGF signal as an oxidizing agent rather than a nitrating agent, cells were treated with VEGF in the presence or absence of the antioxidant and the thiol donor NAC (1 mM). VEGFR2 activation was blocked by NAC (lower panel). These results demonstrate that peroxynitrite-mediated oxidation but not nitration is required to transduce VEGF’s signal. To further test this concept, we compared the functional effects on angiogenesis of FeTPPs, epicatechin, and NAC. Studies of human endothelial cells using the model of capillary morphogenesis in 3-dimensional type I collagen matrix showed that VEGF stimulated the extent of capillary tube formation as indicated by significant increases in tube length and tube area compared with untreated control cultures (Fig. 5B
). VEGF-induced tube formation was completely blocked by FeTPPs and NAC, but was not altered by epicatechin. None of the inhibitors had any effect on the extent or pattern of tube formation in control cultures. Similar effects of FeTPPs in inhibiting endothelial tube formation were also obtained using the potent angiogenic basic fibroblast growth factor (bFGF) in both microvascular and human endothelial cells (data not shown). Several reports indicated that bFGF induces neovascularization indirectly by activation of the VEGF/VEGFR2 system (for review see (28)
.
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VEGF’s angiogenic function requires peroxynitrite-mediated oxidation but not nitration
The above results indicate that VEGF-induced peroxynitrite formation propagates signaling linked to angiogenic responses. The fact that activation of VEGFR2 is enhanced by inhibiting tyrosine nitration but blocked by inhibiting oxidation suggests that the process requires peroxynitrite-mediated oxidation rather than nitration. To further test this concept, we compared the effects on VEGF angiogenic function of the nitration inhibitor with those of oxidation inhibitors, the peroxynitrite decomposition catalyst FeTTPs. As shown in Fig. 6
A, B, VEGF-stimulated proliferation and migration of microvascular endothelial cells were blocked by FeTPPs or the oxidation inhibitor NAC but were not altered by epicatechin. The inhibitors did not alter cell growth or migration in the control cultures. We have confirmed the above results showing that VEGF-stimulated tube formation in microvascular endothelial cells was blocked by FeTPPs and NAC but not by epicatechin (data not shown).
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Peroxynitrite-mediated oxidation is required for retinal neovascularization in vivo
The above results demonstrated the potential role of peroxynitrite in VEGF-mediated signal and function in vitro. VEGF plays a major role in postnatal angiogenesis in a variety of disease models including ischemic retinopathy, psoriasis, and cancer. To further establish the contribution of peroxynitrite to VEGF-mediated angiogenesis in vivo, we analyzed the effects of FeTPPs, epicatechin, or NAC in an established mouse model of ischemic retinal neovascularization (see Materials and Methods). Morphometric analysis of neovascular tufts (summarized in Fig. 7
A) showed that hypoxia-induced retinal neovascularization was almost completely blocked by treatment of animals with FeTPPs (1 mg/kg/day) and NAC (150 mg/kg/day), whereas treatment with epicatechin (10 mg/kg/day) did not prevent neovascularization within the retina. The effects of epicatechin and FeTPPs in blocking tyrosine nitration at the indicated doses were verified using immunolocalization of nitrotyrosine in retinal sections (data not shown). These results lend further support to the hypothesis that VEGF-induced angiogenic signaling involves peroxynitrite-mediated oxidative events.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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2
3
, 29)
. However, our study is the first to identify intracellular peroxynitrite as a signaling molecule that sustains VEGF autophosphorylation and transduces the VEGF signal to downstream targets. Our findings of VEGF-induced simultaneous formation of NO, superoxide, and peroxynitrite support and combine the well-known role of nitric oxide and the recently suggested role of superoxide anion in mediating VEGF’s angiogenic signal in endothelial cells (8
9
10
11
12)
. Peroxynitrite, a nonradical species formed by the interaction between NO and superoxide anion by the fastest reaction known in biology, can cause protein modification by either nitration of tyrosine residues or oxidation of protein-associated thiol groups (30)
. Our findings that peroxynitrite mediates VEGF’s signal and function via thiol oxidation, but not nitration, emphasize the importance of maintaining balanced cellular redox status to mediate physiological NO signaling.
Our results using real-time confocal imaging showed that VEGF stimulation of endothelial cells causes rapid and simultaneous formation of NO, superoxide anion, and peroxynitrite. In good agreement with previous reports, treatment of the cells with the NOS inhibitor (L-NAME) blocked VEGF-induced NO formation while significantly increasing superoxide anion formation (8
9
10
, 31
, 32)
. VEGF-induced intracellular peroxynitrite formation was detected using DCF fluorescence and was compared with a standard curve of serial dilution of exogenous peroxynitrite. VEGF causes DCF fluorescence comparable to 1 µM of peroxynitrite. Our control studies showed that VEGF-induced fluorescence was completely blocked by the peroxynitrite scavenger uric acid (1 mM), FeTPPs (2.5 µM) or by combination of SOD with either L-NAME or catalase, but not by SOD alone or L-NAME alone. This suggests that peroxynitrite is the main source of VEGF-induced DCF fluorescence. VEGF-induced increases in DCF fluorescence have been reported by others, but this effect has not been attributed to peroxynitrite formation (9
, 32
, 33)
. Our functional assays showed that FeTPPs blocks VEGF-induced cell migration, proliferation, and tube formation, indicating a role for peroxynitrite in mediating VEGF’s angiogenic function. FeTPPs is a specific peroxynitrite decomposition catalyst that catalyzes conversion of peroxynitrite into nitrate with a concomitant decrease in the oxidizing and nitrating reactivity of peroxynitrite (34)
. Moreover, treating the cells with a serial dilution of peroxynitrite showed dose-dependent increases in cell proliferation and endothelial alignment, with a maximum effect at 1 µM, which induced responses comparable in timing and magnitude to those seen with VEGF. Moreover, our studies using the chick chorioallantoic membrane model of angiogenesis confirmed our finding that low physiological levels of peroxynitrite mimic VEGF in stimulating angiogenic function in vitro and in vivo.
The angiogenic effects of VEGF in endothelial cells are mediated primarily via binding and autophosphorylation of VEGFR2, followed by association and activation of c-Src kinase (35)
. Therefore, VEGFR2 is a proximal molecular target of peroxynitrite formed on VEGF stimulation. Our results showed that peroxynitrite mimicked the actions of VEGF by causing a time-dependent activation of VEGFR2. VEGF caused VEGFR2 activation within 1 min, which was sustained for up to 30 min. FeTPPs reduced but did not eliminate the immediate VEGF-induced phosphorylation (1 min), but substantially blocked the sustained signal (5–30 min) (Fig. 3A
). A similar pattern was obtained in response to VEGF stimulation in human endothelial cells (Fig. 5A
). The role of peroxynitrite in mediating the VEGF signal was further confirmed by our results showing that inhibiting alone or in combination with SOD blocked VEGF autophosphorylation (Fig. 3B
). In contrast, hydrogen peroxide decomposer (catalase) did not alter the VAGF signal. These results suggest a positive feedback loop in which VEGF triggers autophosphorylation of VEGFR2 to stimulate intracellular peroxynitrite formation, which then sustains the receptor activation and propagates the signal to downstream targets. A potential role of peroxynitrite in causing receptor tyrosine phosphorylation has been reported for both EGF and PDGF receptors (36
, 37)
. However, our study is the first we know to demonstrate the role of peroxynitrite in VEGF receptor activation in vascular endothelial cells.
Peroxynitrite has a dual role of protein modification via nitration of tyrosine residues or oxidation of thiols. We studied the effects of blocking tyrosine nitration by using epicatechin, a constituent of green tea that selectively blocks tyrosine nitration but does not affect thiol oxidation (26
, 27)
. We previously showed that epicatechin (100 µM) prevents tyrosine nitration of PI3-kinase and restores VEGF survival action under conditions of excessive oxidative stress (15)
. Our present studies showed that blocking tyrosine nitration with epicatechin enhanced the tyrosine phosphorylation of both VEGFR2 and c-Src in VEGF- or peroxynitrite-stimulated microvascular endothelial cells (Fig. 4A, B
). A similar pattern was obtained in VEGF-stimulated human endothelial cells (Fig. 5A
). These results exclude the nitrating properties of peroxynitrite in propagating VEGF’s angiogenic signal and point to an oxidation role for peroxynitrite (38
39
40)
in the signal transduction process. The role of peroxynitrite-mediated oxidation in transducing VEGF’s signal is supported by our observation that the cell-permeable oxidation inhibitor and thiol donor NAC blocked VEGF-induced VEGFR2 phosphorylation (Fig. 5A
) and tube formation in human endothelial cells (Fig. 5B
). In addition, functional studies using microvascular endothelial cells showed that VEGF-induced cell growth, migration, and tube formation were blocked by FeTPPs and NAC, but not epicatechin. This supports the concept that peroxynitrite mediates VEGF signaling via oxidation and not nitration. Our results showing the protective effects of NAC in restoring balanced redox to mediate VEGF signal are in good agreement with previous data showing the involvement of oxidative events in transducing the VEGF signal (8
, 41
, 42)
.
VEGF’s role in pathological angiogenesis has been well established in various disease models including cancer, psoriasis, and ischemic retinopathy (43)
. Our recent study using a mouse model of ischemic retinopathy showed that hypoxia induced increases in VEGF expression and retinal neovascularization (24)
. To evaluate the contribution of peroxynitrite in angiogenesis in vivo, we compared the effects of inhibiting peroxynitrite, nitration, or oxidation on VEGF-induced retinal neovascularization. Vascular density analysis showed that decomposing peroxynitrite with FeTPPs or inhibiting oxidation with NAC substantially reduced neovascularization compared with vehicle-treated animals. It is interesting that treatment with epicatechin blocked retinal tyrosine nitration but did not alter hypoxia-induced neovascularization. These results agree well with earlier reports showing that inhibiting peroxynitrite formation blocked retinal neovascularization (44
, 45)
. However, this is the first study to differentiate the dual role of peroxynitrite in mediating oxidative or nitrative events in a disease model. Together with our finding that VEGF-mediated angiogenesis in vitro is blocked by FeTPPs and NAC but enhanced by epicatechin, these observations provide evidence that peroxynitrite is required for VEGF-mediated angiogenesis and that peroxynitrite-mediated protein oxidation, but not nitration, plays an important role. Further studies are in progress to determine the specific role of redox shift and molecular targets of thiol oxidation during VEGF signaling pathway.
In summary, the present study provides compelling evidence that VEGF stimulates endothelial cells to produce peroxynitrite (Fig. 7
B,I). Peroxynitrite serves as a second messenger to activate multiple signaling pathways leading to endothelial migration, proliferation, and tube formation in vitro and angiogenesis in vivo (Fig. 7B,II
). Peroxynitrite fulfills important prerequisites for an intracellular messenger: a small, diffusible, and ubiquitous molecule with a very short half-life. It can be synthesized rapidly in response to external stimuli that induce the formation of nitric oxide and superoxide anion. Thus, even though peroxynitrite does not bind to a receptor, it can change the redox state of the cell. The presented data are consistent with the established roles of NO and superoxide anion in VEGF signaling and provide new insights regarding how the two radicals may contribute to the signaling process. We demonstrated that peroxynitrite causes and sustains tyrosine phosphorylation via oxidation rather than nitration. Taken together, the study suggests that peroxynitrite donors and inhibitors provide potential therapeutic strategies for treatment of angiogenesis-dependent diseases.
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ACKNOWLEDGMENTS |
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Received for publication December 12, 2006. Accepted for publication February 22, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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