Department of Cellular Biology and Anatomy, Department of Ophthalmology, Department of Pharmacology and Toxicology, and Vascular Biology Center, The Medical College of Georgia, Augusta, Georgia 30912
Submitted 16 September 2002 ; accepted in final form 30 April 2003
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ABSTRACT |
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oxygen-induced retinopathy; vaso-obliteration; superoxide; nitric oxide
Alon et al. (2) showed that hyperoxia-induced vasoobliteration occurs via selective apoptosis of retinal vascular endothelial cells. They (2) and other investigators (46) have proposed that premature downregulation of vascular endothelial growth factor (VEGF), an endothelial cell-specific mitogen and survival factor, leads to vaso-obliteration of newly formed capillaries. It also has been shown that hyperoxia causes increased expression of pigment epithelium-derived growth factor (PEDF), a potent angiostatic factor, within the retina (15). A role for direct oxidative damage to endothelial cells by reactive oxygen species (ROS) is supported by studies showing that pretreatment of newborn rats with intravitreal liposome-encapsulated superoxide dismutase (SOD) (41) or systemically administered Trolox (a scavenger of peroxynitrite) (45) diminished the severity of oxygen-induced retinopathy. A role for ROS in ROP is also supported by the fact that antioxidants are in low reserve at early stages of retinal development (42) and that infants with active ROP have low serum levels of reduced glutathione (GSH) (43).
We have shown previously that exposure of neonatal mice to 75% oxygen for 48 h causes degeneration of the retinal capillaries associated with formation of the nitric oxide (NO)-derived oxidant peroxynitrite. Both capillary dropout and protein tyrosine nitration were diminished in mice deficient in endothelial NO synthase (NOS3) or treated with nitro-L-arginine methyl ester (L-NAME), implying that peroxynitrite may be an important mediator of hyperoxia-induced vaso-obliteration (9). Peroxynitrite is a highly reactive oxidant generated by the reaction of NO and superoxide anion. It can be generated directly by NOS, especially when the levels of L-arginine are low (3) or under conditions in which tetrahydbiopterin is oxidized (34). Nucleic acids, lipids, and proteins are all potential targets of peroxynitrite-mediated oxidation reactions (4, 20), and apoptosis is one of the ways in which cells may respond to peroxynitrite-mediated injury (1, 6, 35, 55, 58). It seemed plausible, therefore, that hyperoxia-induced apoptosis of capillary endothelial cells in the developing retina could be mediated in part by the formation of peroxynitrite.
The goal of the present study was to test this hypothesis and to determine the role of peroxynitrite/oxidative stress in endothelial cell apoptosis induced by hyperoxia. The specific aims were to determine whether hyperoxia exposure induces protein tyrosine nitration in cultured endothelial cells, whether this alteration has a role in hyperoxia-mediated endothelial cell death, and whether peroxynitrite exposure alters the intracellular signaling pathway for endothelial cell survival. Our experiments using a tissue culture model system have demonstrated that 40% oxygen induces bovine retinal endothelial cell (BREC) apoptosis as indicated by activation of caspase-3, cleavage of the caspase substrate poly-(ADP-ribose) polymerase (PARP), and increases in numbers of cells with nuclear morphology characteristic of apoptosis. Hyperoxia also increases the formation of NO, superoxide, and peroxynitrite. Pretreatment of BREC with a NOS inhibitor (L-NAME) or with scavengers of superoxide or peroxynitrite blocked the hyperoxia-induced apoptosis. We also have shown that exposing cells to exogenous peroxynitrite blocks growth/survival factor [VEGF, basic fibroblast growth factor (bFGF)]-induced activation of the phosphatidylinositol (PI) 3-kinase/Akt kinase cell survival pathway. Our findings corroborate our in vivo data suggesting a role for NO-derived peroxynitrite as a mediator of hyperoxia-induced apoptosis of capillary endothelial cells in the retina.
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MATERIAL AND METHODS |
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Cell culture. Primary cultures of BREC were prepared as described previously (18). Cells from passages 48 were used in all experiments. Cells were maintained in M199 supplemented with 10% FBS, 10% CS-C complete medium, 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C in a humidified CO2 incubator.
Hyperoxia and normoxia treatment. BREC were grown to 80% confluence and switched to serum-free medium. The next day they were placed in a hyperoxic (40% O2, 5% CO2)or normoxic (21% O2, 5% CO2) environment for 48 h unless otherwise indicated. The 40% O2 level was chosen for hyperoxia exposure on the basis of previous research showing that 40% O2 generates significant oxidative stress without inducing toxicity in retinal epithelial cells and fibroblasts (23). The hyperoxia exposure was performed in a humidified incubator modified by installing the PROOX model 110 oxygen regulator (Reming Bioinstruments, Redfield, NY). The oxygen level was continuously monitored using the PROOX oxygen sensor. The oxygen tension in the culture medium was determined by using a blood gas analyzer (GEM Premier 3000; Instrumentation Laboratory, Lexington, MA) and was found to be 232 ± 11.37 mmHg after 48 h in the 40% O2 environment compared with 164 ± 15.36 mmHg in 21% O2.
Peroxynitrite treatment. Stock concentrations of peroxynitrite were freshly prepared in 0.3 N NaOH. An equal amount of 0.3 N NaOH was used for vehicle control experiments. Decomposed peroxynitrite was used in control experiments and was without effect on any of the parameters analyzed.
Caspase-3 activity. A caspase-3 fluorometric assay kit was used to detect apoptosis. Caspase-3 is an intracellular cysteine protease that exists as a proenzyme and becomes activated during the cascade of events that culminates in apoptosis. BREC in serum-free medium were maintained in 21% O2 (normoxia) or 40% O2 (hyperoxia) for 48 h and then incubated in lysis buffer provided with the kit. Cell lysates were centrifuged at 12,500 g for 10 min. The supernatant was collected, samples equated for protein were reacted with the caspase-3 fluorogenic substrate (DEVD-AFC), and fluorescence was measured with a Cytofluor 4000 spectrophotometer (Framingham, MA) with excitation at 400 nm and emission at 505 nm. The results were expressed as fold increase in caspase-3 activity for the hyperoxia- and normoxia-treated cultures above the level of growing cells maintained for 48 h in media with serum.
Hoechst stain. Apoptotic cells in cultures treated as described above were detected by nuclear staining with Hoechst 33258 (2.5 µg/ml in PBS). Cells were reacted with the stain for 30 min at room temperature, washed twice with PBS, and examined and photographed in a Zeiss Axioskop microscope. At least 15 fields were counted for each treatment. Each field contained over 130 cells. Cells with condensed chromatin or with shrunken, irregular, or fragmented nuclei were considered apoptotic. The number of apoptotic nuclei was calculated relative to the total number of nuclei. Total cell density was also calculated for each treatment condition as an indicator of potential differences in cytotoxicity.
Western blotting analysis. BREC were harvested after various treatments and lysed in modified RIPA buffer (20 mM Tris, pH 7.4, 2.5 mM EDTA, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 40 mM NaF, 10 mM Na4P2O7, and 1 mM PMSF) (5) for 30 min on ice. Insoluble material was removed by centrifugation at 12,000 g at 4°C for 30 min. For analysis of cleaved PARP, NOS1, NOS2, NOS3, phospho-Akt, or Akt, 50 µg of total protein were boiled in 2x Laemmli sample buffer, separated on a 1012% 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 and enhanced chemiluminescence (ECL). Intensity of immunoreactivity was measured using densitometry.
Nitrotyrosine formation. Relative amounts of proteins nitrated on tyrosine were measured using slot-blot techniques as described previously (9, 16). Briefly, cellular protein samples prepared as described above were immobilized onto polyvinylidene difluoride membranes by using a slot blot microfiltration unit (Bio-Rad). A dilution series of peroxynitrite-modified BSA (Cayman Chemical, Ann Harper, MI) was loaded to generate a standard curve, and nitrotyrosine was detected using a monoclonal anti-nitrotyrosine antibody (Cayman Chemical) followed by peroxidase-labeled goat anti-mouse IgG and ECL. Relative levels of nitrotyrosine immunoreactivity were determined by densitometry. Comparison with the standard curve generated from peroxynitrite-modified BSA analysis showed that nitrotyrosine formation in normoxia-treated cells was equivalent to 1.0 µg/ml compared with 1.6 µg/ml nitrosylated BSA in the hyperoxia-treated cells and 4.8 µg/ml nitrosylated BSA in cells treated with 1 mM peroxynitrite.
Immunoprecipitation and Akt kinase assay. BREC plated in 10-cm
dishes were treated with or without peroxynitrite, wortmannin, VEGF, or bFGF
for the desired time. Cell lysates were prepared as described above for
immunoblotting. For immunoprecipitation, 500 µg of protein were incubated
with immobilized Akt 1G1 monoclonal antibody-containing beads overnight at
4°C with gentle rocking. The beads were washed twice with lysis buffer and
kinase buffer (25 mM Tris, pH 7.5, 5 mM -glycerolphosphate, 2 mM DTT,
0.1 mM Na3VO4, and 10 mM MgCl2)
(31) once. Beads were further
incubated in 40 µl of kinase buffer supplemented with 200 µM ATP and 1
µg of glycogen synthase kinase (GSK)-3 fusion protein for 30 min at
30°C. The reaction was terminated with 20 µl of 3x SDS sample
buffer. After boiling and centrifuging, the supernatant was loaded on 12%
SDS-PAGE gel. Western immunoblotting was carried out on the nitrocellulose
membrane with phospho-GSK-3
/
(Ser21/9) antibody as the primary
antibody. Horseradish peroxidase-conjugated goat anti-rabbit antibody and ECL
were used to detect the primary antibody. Intensity of immunoreactivity was
measured using densitometry.
RNA isolation and relative quantitative RT-PCR analysis. Total RNA was isolated from BREC by the guanidine isothiocyanate method, using the RNeasy kit. A total of 2 µg of RNA was reverse transcribed for 60 min at 37°C with 4 units of Omniscript reverse transcriptase, using random primers. One-half microliter of cDNA was added to each PCR reaction, and 18S was used as endogenous standard in PCR reaction. The optimal ratio of 18S:competimers was determined for accurate quantification in the linear range of each RNA. The nucleotide sequences of the oligonucleotide primers used for RT-PCR were selected to span at least one intron-exon splice boundary to eliminate the possibility of amplification of genomic DNA and were as follows: NOS3, 5'-TGCTGCCCAGGACATCTT-3' and 5'-AAGAAGGTGAGAGCCTGG-3'; and NOS2, 5'-ACATGCAGAATGAGTACCGG-3' and 5'-TCAACATCTCCTGGTGGAAC-3'. PCR was performed at 95°C for 15 min, followed by 26 cycles for NOS3 and 32 cycles for NOS2 at 94°C for 1 min, 58°C (NOS3) or 60°C (NOS2) for 30 s, 72°C for 1 min, and then 72°C for 10 min. PCR products were then separated on a 6% acrylamide gel, stained with SYBRE Green, and quantified by densitometry, using the AlphEase imaging analysis system (Alpha Innotech, San Leandro, CA).
Superoxide measurement. Superoxide production was detected using the oxidative fluorescent dye hydroethidine (HE) (39). HE is freely permeable to cells. In the presence of superoxide, HE is oxidized to ethidium bromide (EtBr), which is trapped by intercalating with the DNA (49), resulting in a fluorescence signal that can be quantified by densitometry. Addition of hydrogen peroxide or other oxidants does not significantly increase EtBr fluorescence (10). BREC cultures were treated with either 21% or 40% O2 for 16 h, switched to HEPES buffer (pH 7.4) for 30 min, and then treated with 2 pM HE. The cultures were maintained in the same 21% or 40% O2 environment throughout treatment. After 20 min, the reaction was stopped by switching to fresh HEPES buffer that had been saturated with either 21% or 40% O2. Images were obtained using a laser scanning confocal microscope equipped with a krypton-argon laser. Normoxia- and hyperoxia-treated BREC were processed and imaged in parallel. Laser settings were identical for acquisition of images from both specimens.
Nitrite measurement. A fluorometric assay with the DAN reagent (2,3-diaminonaphthalene) (40) was used to measure the levels of nitrite in the conditioned medium from BREC treated with either 21% or 40% O2. DAN reacts with nitrite under acidic conditions to form 1-(H)-naphthotriazole, a fluorescent product. Briefly, 250 µl of conditioned medium and standards were incubated with 30 µl of DAN reagent (633 µM in 0.67 N HCl) at room temperature in the dark for 15 min. The reaction was stopped by adding 30 µl of 2 N NaOH, and fluorescence was measured using the Cytofluor 4000 spectrophotometer with excitation at 360 nm and emission at 409 nm. Nitrite concentrations in the different samples were calculated using a standard curve generated from a nitrite dilution series of 10 to 0.156 µM.
Statistical analysis. Each experiment was repeated at least three times. Results are expressed as means ± SE. Statistical significance was determined by one- or two-way ANOVA. P values <0.05 were considered significant.
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RESULTS |
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Hyperoxia increases formation of peroxynitrite, NO, and superoxide. Peroxynitrite is a short-lived molecule at physiological pH, but it produces stable nitration of protein tyrosine residues. Thus nitrotyrosine immunoreactivity can be used to assay for peroxynitrite formation (22, 25, 50). Slot-blot analysis of proteins from BREC with anti-nitrotyrosine antibody revealed an increase of 6070% in the amount of nitrotyrosine in hyperoxia-treated cells over that found in normoxic controls (Fig. 2). This finding suggests that hyperoxia causes a significant increase in the formation of peroxynitrite.
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To further evaluate the effects of hyperoxia on formation of reactive nitrogen species, we used quantitative fluorophotometric assay to measure the accumulation of nitrite in the tissue culture media. Increases in media nitrite accumulation are an indicator of NOS activity in forming NO. Nitrite may also accumulate as a breakdown product of peroxynitrite. Our analyses showed a 1.5-fold increase in nitrite levels in the medium from BREC exposed to 40% O2 for 48 h compared with the medium from normoxic controls (Fig. 3).
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To see whether the increases in peroxynitrite and nitrite formation were associated with increases in NOS protein expression, we determined the effects of hyperoxia on the expression of different isoforms of NOS after 48 h. Western blot analysis showed a slight increase in NOS3 protein levels in some of the hyperoxia-exposed cultures, but the group differences were not statistically significant (Fig. 3A). Neither NOS1 nor NOS2 was detected in either normoxia- or hyperoxia-treated cultures.
We next evaluated the effects of hyperoxia on expression of NOS mRNA expression. For these analyses, we studied BREC exposed to hyperoxia for 16 h. We chose this time period because we wanted to avoid potential effects of apoptosis on the results. We also reasoned that if increases in NOS expression contribute to increases in formation of NO and peroxynitrite, increases in mRNA levels should occur relatively early during the hyperoxia exposure. However, this analysis showed only a slight increase in NOS3 mRNA compared with normoxic controls (P < 0.05, Fig. 4). There was no change in immunologic NOS (NOS2) expression. Neuronal NOS (NOS1) was not detected. These results suggest that hyperoxia increases NO formation in BREC due to an action increasing NOS3 activity, with little or no effect on NOS expression.
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Because peroxynitrite is a combination product of nitric oxide and superoxide anion, we determined the formation of superoxide anion after hyperoxia exposure. This analysis was also done in cells exposed to hyperoxia for 16 h because DNA fragmentation during apoptosis can alter the HE staining reaction. BREC exposed to 40% O2 for 16 h showed a 3.4-fold increase (P < 0.05) in EtBr fluorescence compared with normoxic controls. This effect was blocked when cells were preincubated with polyethylene glycol-superoxide dismutase (PEG-SOD; 100 U/ml), demonstrating the specificity of the reaction for superoxide anion (Fig. 5).
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Blocking NOS, peroxynitrite, or superoxide inhibits hyperoxia-induced apoptosis. Treatment of BREC with the NOS inhibitor L-NAME (0.5 µM), the superoxide scavenger PEG-SOD (100 U/ml), or the peroxynitrite scavenger uric acid (1 mM) before (30 min) and throughout exposure to hyperoxia (40% O2, 48 h) completely inhibited the increase in apoptosis (Fig. 6). Uric acid binds peroxynitrite but not NO and inhibits the action of peroxynitrite in causing tyrosine nitration (24, 56). Cell counts confirmed that hyperoxia caused a significant decrease in cell density (from 148 ± 6 cells/field in normoxic cultures to 133 ± 18 cells/field in hyperoxic cultures). This decrease in cell number was completely prevented by the inhibitor treatments. Together, these data strongly suggest that the hyperoxia-induced apoptosis in BREC occurs via a peroxynitrite-mediated mechanism.
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Peroxynitrite alters the PI 3-kinase/Akt kinase signal transduction pathway for endothelial cell survival. Cell growth and cell death are highly regulated processes required for normal vascular development. Increasing evidence indicates that activation of PI 3-kinase, and its downstream target Akt kinase, are involved in antiapoptotic signaling induced by growth/survival factors (27, 33, 44). Because peroxynitrite can inactivate tyrosine kinases by nitration of key tyrosine residues, we hypothesized that peroxynitrite might cause BREC apoptosis by impairing key survival pathways activated by growth/survival factors. To test this possibility, we evaluated the ability of VEGF or bFGF to activate the PI 3-kinase/Akt kinase pathway in BREC treated with peroxynitrite.
As expected, VEGF and bFGF increased the level of phospho-Akt as well as Akt kinase activity in normoxic BREC (Figs. 7 and 8). These stimulatory effects of VEGF and bFGF were substantially inhibited by pretreatment of the cultures with peroxynitrite. Treatment with peroxynitrite alone increased levels of phospho-Akt and Akt kinase activity, though to a lesser extent than either VEGF or bFGF. Wortmannin, a specific PI 3-kinase inhibitor, effectively blocked Akt kinase activation (Fig. 9). The data in Figs. 7 and 8 suggest that peroxynitrite has the capability of interfering with VEGF- and bFGF-induced activation of the PI 3-kinase/Akt kinase transduction pathway, providing a plausible mechanism whereby peroxynitrite generated during prolonged hyperoxia could lead to BREC apoptosis.
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DISCUSSION |
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Our studies provide evidence that peroxynitrite formed under hyperoxic conditions may be an important mediator of the apoptosis. Specifically, hyperoxia caused an increase in the formation of NO and superoxide. This in turn caused an increase in endothelial cell peroxynitrite, as indicated by increased levels of nitrotyrosine. Although other reactive nitrogen species can produce nitration of tyrosine residues, nitrotyrosine is considered a likely marker of peroxynitrite under conditions of simultaneous production of NO and superoxide (22, 25, 50). In addition, experiments using pharmacological inhibitors to scavenge superoxide or peroxynitrite or to inhibit the activity of NOS all showed a marked reduction in BREC apoptosis. This finding provides further support for the role of peroxynitrite formation in hyperoxia-induced endothelial cell apoptosis. NOS1 and NOS2 proteins were not detected in BREC, and NOS3 protein levels were not altered by hyperoxia exposure. Thus it is considered likely that the hyperoxia-induced increase in NO formation is the result of an increase in NOS3 activity. Hyperoxia-induced activation of NOS in the absence of increases in NOS protein has been observed in rat cerebral cortex (53).
Other investigators (14) have reported that hyperoxia inhibits the proliferation of retinal vascular endothelial cells but not pericytes. Their experiments showed that the endothelial cells failed to recover their proliferative ability after being returned to the room air environment. Although their study showed no evidence of cytotoxic cell injury, cytotoxicity assays do not detect cell death by apoptosis. Thus it is possible that the inhibition of endothelial cell growth they observed actually involved an increase in apoptosis.
Activation of the caspase family of cysteine proteases has been demonstrated to be a central event in apoptosis (13, 21). Caspase-3 is considered to be one of the major downstream caspases of the apoptotic cascade and has been shown to play a critical role in apoptosis induced by a range of stimuli in many cell types, including endothelial cells exposed to peroxynitrite-modified oxidized low-density lipoprotein (LDL) (32). Our results showed that hyperoxia caused significant increases in activity of caspase-3 and in the amount of cleaved PARP, the caspase-3 substrate, as well as in cells with nuclear morphology characteristic of apoptosis. These data are consistent with the hypothesis that exposure of endothelial cells to chronic, mild hyperoxia results in acceleration of apoptosis through a cascade of signaling events resulting in caspase-3 activation and PARP cleavage.
Cell death via apoptosis occurs as a response to oxidative cellular damage in many cell types (12, 19, 51, 57). Peroxynitrite is known to be a much more potent and reactive oxidant than its constituents, NO and superoxide. Although cultured endothelial cells are normally highly resistant to apoptosis (47, 48), peroxynitrite-oxidized LDL has been shown to induce apoptosis of endothelial cells from the aorta and umbilical vein (32). Peroxynitrite-mediated alterations have also been shown to induce apoptosis of vascular endothelial cells in vivo in stroke-prone hypertensive rats (36). The mechanisms of peroxynitrite-induced apoptosis remain unclear. Many growth factors have been shown to prevent apoptosis and promote cell survival through activation of the PI 3-kinase/Akt kinase pathway. Our data demonstrate that peroxynitrite is capable of blocking activation of the PI 3-kinase/Akt kinase pathway in BREC by VEGF and bFGF. Both of these factors are known to be key growth/survival factors in the retina (7, 26, 28, 46, 52). It is possible, therefore, that the pathological effects of peroxynitrite during the vaso-obliteration phase of oxygen-induced retinopathy could be due in part to interference with VEGF- and/or bFGF-mediated survival functions.
Further studies are needed to determine the molecular mechanisms by which peroxynitrite modifies specific signaling pathways. It is known, however, that peroxynitrite can modify tyrosine-containing proteins, such as protein kinase C (30), Mn-SOD (38), and c-SRC tyrosine kinase (37). This modification can inactivate the tyrosine kinase activity of the molecules, blocking their ability to effectively function in signal transduction (8). Peroxynitrite has also been found to induce tyrosine phosphorylation in some experimental settings, and its effect in activating the PI 3-kinase/Akt pathway is well established (for review, see Ref. 29). Our data showing that peroxynitrite pretreatment blocks the effects of VEGF or bFGF in activating the PI 3-kinase/Akt pathway suggest that peroxynitrite also initiates other signaling processes that interfere with the signaling pathway that mediates cell survival. This concept is further supported by our studies of high-glucose-mediated oxidative stress, which have shown that chronic exposure to high glucose or overnight treatment with 100 µM peroxynitrite causes accelerated apoptosis in BREC (17). This effect is accompanied by increases in activity of apoptosis-associated stress protein p38 mitogen-activated protein kinase, whereas the effects of exogenous VEGF in increasing Akt activation and promoting cell survival are inhibited, indicating that cell survival signaling function is impaired.
In summary, our studies suggest that the NO-derived oxidant peroxynitrite plays a key role in hyperoxia-induced retinal capillary endothelial cell apoptosis, possibly by blocking growth factor-induced activation of the PI 3-kinase/Akt kinase survival pathway. Our studies also suggest that effective blockade of peroxynitrite formation may prevent oxygen-induced endothelial cell apoptosis. These data may have important clinical implications for the management of infants at risk for retinopathy of prematurity.
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DISCLOSURES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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