Division of Pulmonary and Critical Care Medicine, Department of Medicine, Johns Hopkins School of Medicine, Baltimore, Maryland
Submitted 7 December 2004 ; accepted in final form 15 June 2005
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ABSTRACT |
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pulmonary hypertension
Most mammalian systems possess several mechanisms by which pHi homeostasis is maintained. These include the Na+-HCO3 cotransporter, Na+-dependent Cl/HCO3 exchange, Na+-independent Cl/HCO3 exchange, and Na+/H+ exchange (NHE). All of these exchangers have been shown to exist in vascular smooth muscle (1, 20, 27, 33, 35), and, in PASMCs, Na+/H+ has been demonstrated to play a significant role in the regulation of resting pHi (35). Na+/H+ exchangers reside in the plasma membrane and use the transmembrane Na+ gradient to extrude protons. Genes have been identified that encode at least nine isoforms of the Na+/H+ exchanger (NHE19), with NHE13 having been best characterized. NHE1 is ubiquitously expressed, whereas NHE2 and NHE3 are found predominately in the kidney and gastrointestinal epithelium (7, 31, 40, 43), although low-level expression of both isoforms in the lung has been reported (7, 43). Little is known about the function of NHE4 and NHE5, although their expression is restricted primarily to the gastrointestinal tract and brain, respectively (3, 30). Very low expression of NHE69 has been reported in whole lung tissue, but localization of these isoforms is restricted to subcellular organelles (12, 23, 26, 28, 29). The cell-specific expression of NHEs in the pulmonary vasculature has not been studied, although cultured pulmonary endothelial cells have been shown to possess only NHE1 (8).
Many stimuli, including acute hypoxia and growth factors, induce PASMC alkalinization (20, 33). The alkaline shift in pHi observed in response to platelet-derived growth factor and epidermal growth factor occurs due to activation of NHE activity, whereas inhibition of NHE prevents proliferation in response to growth factors (33). These results indicate a functional role for pHi and NHE in PASMC growth. Furthermore, inhibition of NHE with amiloride analogs attenuated vascular remodeling and pulmonary hypertension in rats exposed to chronic hypoxia (34). The results from these studies suggest that both hypoxia and growth factors can modulate pHi and that PASMC growth and subsequent vascular remodeling during development of hypoxic pulmonary hypertension may be due to alterations in NHE activity and pHi. However, the effect of chronic hypoxia on basal pHi and Na+/H+ exchanger activity is not known. Thus, in this study, we used fluorescent microscopy to measure basal pHi and Na+/H+ exchanger activity, and RT-PCR and immunoblot techniques to examine NHE expression in pulmonary vascular smooth muscle isolated from normoxic and chronically hypoxic mice.
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METHODS |
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All procedures were approved by the Animal Care and Use Committee of The Johns Hopkins University School of Medicine. Male C57B6 mice were placed in a hypoxic chamber for 21 days as previously described (39). The chamber was continuously flushed with a mixture of room air and N2 (10 ± 0.5% O2) to maintain low CO2 concentrations (<0.5%). Chamber O2 and CO2 concentrations were continuously monitored (ProOx 110 oxygen analyzer; Biospherix, Redfield, NY and LB-2 CO2 analyzer; Sensormedics, Anaheim, CA). The mice were exposed to room air for 10 min twice a week to clean the cages and replenish food and water supplies. Normoxic controls were kept in the room air next to the hypoxia exposure chamber. At the end of exposure, mice were injected with heparin and anesthetized with pentobarbital sodium (43 mg/kg ip). The heart and lungs were removed en bloc and transferred to a petri dish of HEPES-buffered salt solution (HBSS) containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.2 with 5 M NaOH. After removal of the atria, the right ventricle (RV) of the heart was separated from the left ventricle and the septum (LV+S), and the two portions were blotted dry and weighed.
Cell Isolation and Culture
The method for obtaining single PASMCs has been described previously (39). Briefly, intrapulmonary arteries (100400 µm outer diameter) were isolated and cleaned of connective tissue. After disrupting the endothelium by gently rubbing the luminal surface with a cotton swab, we allowed the arteries to recover for 30 min in cold (4°C) HBSS, followed by 20 min in reduced-Ca2+ (20 µM CaCl2) HBSS at room temperature. The tissue was digested in reduced-Ca2+ HBSS containing collagenase (type I, 1,750 U/ml), papain (9.5 U/ml), bovine serum albumin (2 mg/ml), and DTT (1 mM) at 37°C for 10 min. After digestion, single smooth muscle cells were dispersed by gentle trituration with a wide-bore transfer pipette in Ca2+-free HBSS, and the cell suspension was placed on 25-mm glass coverslips. PASMCs were cultured under normoxic conditions in SmBm complete media supplemented with 10% FCS for 34 days and placed in serum-free media 24 h before experiments.
pHi Measurements
PASMCs were placed in a laminar flow cell chamber perfused with HBSS with pH adjusted to 7.4. pHi was measured in cells incubated with the membrane-permeant (acetoxymethyl ester) form of the pH-sensitive fluorescent dye BCECF-AM for 60 min at 37°C under an atmosphere of 21% O2-5% CO2. Cells were then washed with HBSS for 15 min at 37°C to remove extracellular dye and allow complete deesterification of cytosolic dye. Ratiometric measurement of fluorescence from the dyes was performed on a workstation (Intracellular Imaging, Cincinnati, OH) consisting of a Nikon TSE 100 Ellipse inverted microscope with epifluorescence attachments. The light beams from a xenon arc lamp were filtered by interference filters at 490 and 440 nm and focused onto the PASMCs under examination via a x20 fluorescence objective (Super Fluor 20, Nikon). Light emitted from the cell at 530 nm was returned through the objective and detected by a cooled charge-coupled device imaging camera. An electronic shutter (Sutter Instruments) was used to minimize photobleaching of dye. Protocols were executed and data were collected online with InCyte software (Intracellular Imaging). pHi was estimated from in situ calibration after each experiment. Cells were perfused with a solution containing (in mM) 105 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, 20 HEPES-Tris, and 0.01 nigericin to allow pHi to equilibrate to external pH (42). A two-point calibration was created from fluorescence measured as pHi was adjusted with KOH from 6.5 to 7.5. Intracellular H+ concentration ([H+]i) was determined from pHi using the formula pHi = log [H+]i.
RT-PCR
Total RNA was prepared from intralobar pulmonary arteries by TRIzol extraction. Two arteries each from three mice were isolated and combined per sample after the endothelium was denuded by gently rubbing the lumen with a cotton swab. We have previously demonstrated complete endothelial cell disruption using this technique (38). Isolated total RNA was dissolved in 40 µl of diethyl pyrocarbonate water and stored at 80°C until use. Reverse transcription was performed using the First-strand cDNA synthesis kit (Pharmacia Biotech). Three micrograms of the total RNA were reverse transcribed using random hexamers incubated for 1 h at 37°C. The reverse transcriptase was inactivated by heating the mixture for 5 min at 90°C. Specific primers for NHE1, NHE2, NHE3, and -actin (Table 1) were designed from sequences of the coding regions corresponding to mouse NHE13 and
-actin genes (13, 31, 42). PCR was performed using the GeneAmp PCR system 2700 (Applied Biosystems) using Taq polymerase. Four microliters of the first-strand cDNA mixture were amplified by denaturing at 94°C for 1 min, annealing at 60°C for 1 min, and extending at 72°C for 1.5 min. Preliminary experiments were performed to determine the optimum number of cycles (35 cycles). After 35 cycles, a final extension was run at 72°C for 10 min. PCR products were electrophoresed through a 1% agarose gel and stained with ethidium bromide for visualization under UV light. Quantification was performed by densitometry.
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For each sample, cells from three animals were isolated and grown in 60-mm petri dishes containing SmBm complete media supplemented with 10% FCS for 35 days and then serum starved for 24 h before harvest. PASMCs were scraped and cells were lysed in 1 ml of cold lysis buffer containing (in mM) 25 HEPES, 1 DTT, and the protease inhibitor cocktail Complete tablets (Roche). Protein concentrations were calculated from a standard Bradford assay. For each sample, 10 µg of total protein were used in each lane, separated on 10% Tris·HCl SDS-PAGE gels, and transferred to nitrocellulose membranes. Membranes were blocked in 5% nonfat milk for 2 h, probed with primary antibody (Chemicon) overnight at 4°C, washed, and incubated in secondary goat anti-rabbit antibody (Bio-Rad) for 1 h. Bands were visualized by enhanced chemiluminescence. Membranes were then stripped and reprobed for -actin. Densitometry was performed to quantify the amount of protein, and the ratio of NHE1 to
-actin was calculated. Fold induction was determined by setting the ratio of NHE1/
-actin in normoxic animals equal to 1.
Experimental Protocols
Effect of chronic hypoxia and EIPA on basal pHi. Baseline pHi was measured for 3 min in PASMCs from normoxic and chronically hypoxic mice. Values were averaged to obtain a mean value for each cell. To assess the role of HCO3 in maintenance of basal pHi, some cells were initially perfused with a modified Krebs bicarbonate (KRB) solution containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 25 NaHCO3, 1.1 glucose, and 1.2 KH2PO4 and gassed with 16% O2 and 5% CO2 to maintain pH at 7.4. Baseline pHi was measured for 3 min, after which the cells were perfused with HBSS and pHi was measured for an additional 5 min. The effect of EIPA, an NHE inhibitor, on baseline pHi was determined by monitoring pHi for 3 min before and 5 min after exposure to 10 µM EIPA. These experiments were performed in cells perfused with both HCO3-containing (KRB) and HCO3-free (HBSS) solutions.
Effect of chronic hypoxia on NHE. A standard ammonia pulse technique was used to measure NHE activity (see Fig. 3). PASMCs loaded with BCECF were placed on the fluorescence microscope and perfused at a rate of 2.5 ml/min with HEPES1 solution containing (in mM) 130 NaCl, 5 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, and 20 HEPES, with pH adjusted to 7.4 with NaOH at 37°C. Baseline pHi was measured for 2 min before cells were briefly exposed to NH4Cl (ammonium pulse) by perfusing with HEPES2 solution containing (in mM) 110 NaCl, 20 NH4Cl, 5 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, and 20 HEPES at a pH of 7.4 using NaOH for 3 min. The ammonium pulse caused alkalinization due to influx of NH3 and buffering of intracellular H+. Washout of NH4Cl in the absence of extracellular Na+ using a Na+ and NH4+-free solution containing (in mM) 130 choline chloride, 5 KCl, 1 MgCl2, 1.5 CaCl2, 10 glucose, and 20 HEPES at a pH of 7.4 using KOH for 10 min results in acidification due to rapid diffusion and washout of NH3, leaving behind H+. The external solution was then switched back to HEPES1 solution for 10 min. Readdition of extracellular Na+ allows activation of NHE and recovery from acidification to basal levels. The rate of Na+-dependent recovery from intracellular acidification corresponds to NHE activity.
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All values are expressed as means ± SE; n refers to the number of cells, hearts, or arteries tested. In each experiment, cells or arteries were used from a minimum of three different animals. Data were compared using Student's t-test (unpaired) or two-way ANOVA with Tukey's or Bonferroni's posttest, as applicable. P < 0.05 was accepted as statistically significant.
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RESULTS |
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To verify the development of pulmonary hypertension in chronically hypoxic animals, the degree of right ventricular hypertrophy was determined by measuring RV weight, LV+S weight, and the ratio of RV/LV+S weights (Fig. 1). Mice exposed to chronic hypoxia (n = 36) had significantly higher RV weights than normoxic mice (n = 31). LV+S weights were similar in both groups, leading to a higher RV/LV+S ratio in chronically hypoxic mice due to RV hypertrophy. These results indicate that our mice developed significant pulmonary hypertension during hypoxic exposure and are consistent with previous results (39).
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Basal pHi was monitored for 3 min in PASMCs isolated from normoxic and chronically hypoxic mice, and an average value for each cell was determined (Fig. 2). When cells were perfused with a bicarbonate-containing solution, basal pHi was 7.34 ± 0.03 (n = 18 cells from 3 animals), corresponding to an [H+]i of 0.0479 ± 0.003 µM. Basal pHi was more acidic (6.88 ± 0.01), and [H+]i was greater (0.1,453 ± 0.006 µM), when measured in PASMCs perfused with HEPES-buffered solution (n = 131 cells from 9 animals). Basal pHi was increased, and [H+]i reduced, in cells isolated from chronically hypoxic mice and perfused with either bicarbonate-containing (7.59 ± 0.04 and 0.0276 ± 0.006 µM; n = 13 cells from 3 animals) or HEPES-buffered (7.23 ± 0.04 and 0.0772 ± 0.008 µM; n = 64 cells from 5 animals) extracellular solution.
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NHE activity, measured as the Na+-dependent rate of recovery from ammonium-induced acidosis, was compared in PASMCs isolated from normoxic (n = 36 cells from 4 animals) and chronically hypoxic (n = 25 cells from 3 animals) mice (Fig. 3). PASMCs isolated from chronically hypoxic mice exhibited significantly faster recovery rates (0.201 ± 0.02 vs. 0.126 ± 0.02 pH U/min). Moreover, the maximum pHi to which cells recovered was also significantly higher in PASMCs isolated from chronically hypoxic mice (7.00 ± 0.04 vs. 6.73 ± 0.06).
Effect of NHE Inhibition on Basal pHi and NHE Activity
Addition of EIPA (105 M), an inhibitor of NHE, reduced basal pHi in cells isolated from normoxic animals perfused with either bicarbonate-containing or bicarbonate-free extracellular solution (Fig. 4). In cells isolated from chronically hypoxic animals, pHi decreased from 7.59 ± 0.04 to 7.36 ± 0.03 (n = 13 from 3 animals) in cells perfused with bicarbonate-containing extracellular solution and from 7.23 ± 0.05 to 6.56 ± 0.02 (n = 25 from 3 animals) in cells perfused with bicarbonate-free extracellular solution.
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Identification of NHE Isoforms in PASMCs
The NHE isoforms responsible for NHE in pulmonary vascular smooth muscle have not been determined. To date, nine isoforms of NHE have been identified. Because NHE13 exhibit the widest tissue distribution, NHE45 were not present in whole lung tissue (3, 30), and localization of NHE69 is restricted to subcellular organelles (23, 26, 28, 29), we focused on NHE13 in PASMCs. With the use of RT-PCR with primers specific for mouse NHE13, the presence of NHE1, NHE2, and NHE3 was demonstrated in mouse kidney under normoxic conditions (Fig. 5), results consistent with previous observations (40, 43). In mouse PASMCs, however, only NHE1 was expressed, with no visible PCR product for NHE2 or NHE3 observed. The identity of our PCR product as NHE1 was confirmed by sequencing the band excised from the gel. The presence of NHE1 protein in mouse pulmonary vascular smooth muscle was confirmed via immunoblot (Fig. 6).
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The effect of exposure to chronic hypoxia on NHE1 expression in mouse pulmonary vascular smooth muscle was determined by RT-PCR performed on RNA extracted from endothelium-denuded intrapulmonary arteries freshly isolated from normoxic and chronically hypoxic mice. This preparation was used to limit the effects of reoxygenation and provides a sample that is 90% smooth muscle. After exposure to chronic hypoxia, NHE1 gene expression increased significantly, whereas hypoxia had no effect on the expression of -actin (Fig. 6). Similarly, Western blot analysis indicated that the hypoxia-induced increase in NHE1 gene expression correlated with increased NHE1 protein expression in PASMCs isolated from chronically hypoxic mice.
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DISCUSSION |
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There are four cellular transporters responsible for regulating pHi: Na+-HCO3 cotransport, Na+-dependent Cl/HCO3 exchange, Na+-independent Cl/HCO3 exchange, and NHE. All of these exchangers appear to be active in PASMCs under basal conditions (11, 20, 35). In our cells, in the presence of bicarbonate, baseline pHi was similar to that measured in bovine and guinea pig PASMCs (33, 35) and substantially higher than that measured in cat PASMCs (20). Removal of extracellular bicarbonate reduced pHi, suggesting that either Na+-HCO3 cotransport or Na+-dependent Cl/HCO3 exchanger, both of which mediate net acid efflux (15, 20, 27), helped to maintain a more alkaline basal pHi. A similar drop in pHi in the absence of HCO3 was observed in bovine and feline PASMCs (20, 33), whereas guinea pig PASMCs exhibited an increase in pHi with removal of bicarbonate (35). The values for pHi measured in the absence of HCO3 in our cells correlate well with values measured in ferret and cat PASMCs (11, 20) but are somewhat lower than those measured in bovine and guinea pig (33, 35). The variations in basal pHi and effect of bicarbonate removal between studies are not completely understood and may be due to differences in species, conditions of the experiments, or measurement techniques.
While a reduction in basal pHi in the absence of HCO3 indicates a role for Na+-HCO3 cotransport or Cl/HCO3 exchangers in regulating resting pHi, a significant decrease in pHi in the presence of EIPA, an NHE inhibitor, also demonstrates a role for NHE in PASMC pHi homeostasis. The decrease in pHi was observed in PASMCs perfused with either HCO3-containing or HCO3-free solutions, indicating that activation of the exchanger is not restricted to conditions of acid load (i.e., in the absence of HCO3). These results confirm previous observations in ferret, cat, bovine, and guinea pig PASMCs (11, 20, 33, 35).
Exposure to chronic hypoxia resulted in a significant increase in basal pHi in PASMCs. Our results are consistent with previous studies that indicated acute hypoxia increased pHi in feline PASMCs (20). Our chronic hypoxic studies indicate a larger change in basal pHi than the acutely hypoxic trials in the cat PASMCs, which may be the result of different mechanisms that induce changes in pHi for acute and chronic hypoxic conditions. With acute hypoxia, the alkalinization of PASMCs was found to be due to altered activity of Cl/HCO3 exchange (20). In the case of chronic hypoxia, the magnitude of the increase was similar in PASMCs perfused with either HCO3-containing or HCO3-free solutions, suggesting that the majority of the increase in pHi was due to increased H+ efflux secondary to activation of NHE activity and not due to alterations in Cl/HCO3 exchanger activity. Enhanced NHE activity was verified using the ammonium pulse technique, which demonstrated a doubling in the rate of Na+-dependent recovery from acidosis under HCO3-free conditions. The increase in the rate of recovery could be blocked completely in the presence of EIPA, further confirming the role of NHE. Finally, basal pHi in PASMCs isolated from chronically hypoxic rats exhibited a marked reduction in resting pHi when exposed to EIPA. These data indicate that exposure to chronic hypoxic causes a marked alkalinization in PASMCs due to increased NHE activity and H+ efflux.
An increase in NHE activity could be achieved by either enhanced activation or an increase in exchanger expression. Activation of the exchanger may be accomplished by phosphorylation secondary to activation of PKC or MAPK (5, 9). However, long-term adaptation to hypoxia, as occurs over 3 wk, generally involves changes in gene expression. The fact that the changes in pHi and NHE activity were observed for several days after reexposure to normoxia (during culture) also suggests that regulation of exchanger activity via phosphorylation, which is rapidly reversible, plays a minimal role. Thus we concluded that chronic exposure to hypoxia may induce alkalinization via increased NHE1 gene transcription.
To test this hypothesis, we examined NHE expression in our cells. Although nine genes have been identified that encode different isoforms of the NHE exchanger (NHE19), and the lung has been shown to express both NHE13 (7, 43), the cell-specific expression of NHE isoforms in the pulmonary vasculature has not been studied. Using specific primers for NHE13, we found, via RT-PCR, that only NHE1 was expressed in mouse PASMCs. The lack of expression for NHE23 was not due to erroneous primers or PCR conditions, as products for NHE2 and NHE3 were readily observed in mouse kidney samples, and is consistent with results obtained in aortic smooth muscle (19).
After exposure to chronic hypoxia, RT-PCR analysis revealed that the levels of NHE1 expression were markedly increased, indicating increased gene transcription. The hypoxia-induced upregulation of NHE1 gene expression was associated with an increase in NHE1 protein expression, as determined via immunoblot performed on PASMCs isolated from both normoxic and chronically hypoxic mice. These results suggest that the increase in NHE activity and corresponding alkaline pHi measured in PASMCs from chronically hypoxic mice were due to increased expression of NHE1. Moreover, since the increase in NHE1 expression was measured in pulmonary arteries freshly isolated from hypoxic animals, these results also suggest that it is unlikely the changes in pHi and NHE activity measured in cells from chronically hypoxic mice were an artifact of reoxygenation during the culture period.
Very little is known about the regulation of NHE1 expression by hypoxia. NHE1 gene expression was found to be unchanged in endothelial cells cultured under hypoxic conditions for 72 h (8), whereas intermittent hypoxia caused acidification and a reduction in NHE1 expression in the brain and central nervous system (CNS) (10). It is unclear whether the lack of effect of induction of NHE1 expression by hypoxia in endothelial cells and CNS was due to cell specificity of the response, level, duration, or mode of hypoxic exposure. It is also not clear whether the hypoxia-induced upregulation of NHE1 expression was a direct effect of hypoxia per se or due to a change in circulating factors, mechanical influences due to increased arterial pressure, or other factors. For example, growth factors, the activity and amount of which may be elevated in animals exposed to chronic hypoxia, have been shown to participate in the regulation of NHE1 expression (4, 37). Thus the exact mechanisms by which chronic hypoxia caused increased NHE1 expression in PASMCs remain to be determined.
Regulation of NHE and pHi is vital for maintaining cell viability. pHi modulates a number of important cell functions, including volume regulation, signal transduction pathways involved in cell proliferation, and mediator release (8, 32). A role for alkaline pHi in hypoxic regulation of vascular caliber is indicated by studies demonstrating that 1) acute hypoxia induces an increase in pulmonary vascular smooth muscle pHi (20), 2) late hypoxic contraction is accompanied by an increase in pHi (18), 3) activation of NHE and alkalinization is required for PASMC proliferation in response to growth factors (33), and 4) pulmonary vascular remodeling during chronic hypoxia can be prevented by inhibiting NHE (34). In addition to facilitating vascular remodeling, the role of pHi in development of hypoxic pulmonary hypertension may also be related to its ability to influence smooth muscle cell function. For example, alkaline pHi reduces voltage-gated K+ channel activity (2), induces Ca2+ influx (11, 14, 25), and causes contraction (16) in pulmonary vascular smooth muscle. Indeed, the increase in PASMC pHi in response to acute hypoxia was accompanied by an increase in [Ca2+]i (14, 41).
In summary, we found that basal pHi was more alkaline in PASMCs isolated from mice exposed to chronic hypoxia via an increase in NHE1 expression. Although it is not clear whether the observed change in pHi alone is sufficient to induce PASMC proliferation, the hypoxia-induced alkalinization could be permissive for cell growth, contributing to the development of the vascular remodeling characteristic of hypoxic pulmonary hypertension.
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GRANTS |
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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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REFERENCES |
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