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Hypertension. 2006;48:497-503
Published online before print July 24, 2006, doi: 10.1161/01.HYP.0000235865.03528.f1
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(Hypertension. 2006;48:497.)
© 2006 American Heart Association, Inc.

Original Articles

Tumor Necrosis Factor-{alpha} and Vascular Angiotensin II in Estrogen-Deficient Rats

Ivan A. Arenas; Stephen J. Armstrong; Yi Xu; Sandra T. Davidge

From the Perinatal Research Center, Departments of Obstetrics and Gynecology and Physiology, University of Alberta, Edmonton, Alberta, Canada.

Correspondence to Sandra T. Davidge, Perinatal Research Centre, 220 Heritage Medical Research Center, University of Alberta, Edmonton, Alberta, Canada T6G 2S2. E-mail sandra.davidge{at}ualberta.ca


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Alterations in the vascular angiotensin II system may play a role in the pathophysiology of vascular disease after menopause. In previous studies we have shown that an increase in tumor necrosis factor (TNF)-{alpha} levels in aging rats because of estrogen deficiency may result in vascular dysfunction. In this study we investigated the effect of TNF-{alpha} inhibition in angiotensin II modulation of vascular function in aging female animals. Female rats approaching reproductive senescence (12 to 15 months old) were ovariectomized and treated with placebo, estrogen, or a selective TNF-{alpha} inhibitor (etanercept) for 4 weeks. Expression of angiotensin II in mesenteric arteries was evaluated by immunofluorescence, and the expression of angiotensin-converting enzyme and angiotensin type I receptor (AT1R) was investigated by Western immunoblot. Vascular function was assessed in mesenteric arteries using the myograph system, and the role of endogenous angiotensin II on adrenergic vasoconstriction was evaluated in vitro by selective AT1R blockade (Candesartan; 10 µmol/L). Our data demonstrate that estrogen-depleted rats have higher serum levels of TNF-{alpha} and greater sensitivity to phenylephrine vasoconstriction compared with estrogen-replaced animals, which was attenuated by AT1R blockade. In vivo TNF-{alpha} inhibition or estrogen replacement reduced phenylephrine constriction of mesenteric arteries and decreased the modulation of this vasoconstriction by candesartan. These functional changes were accompanied by a reduction in the vascular expression of angiotensin II, angiotensin-converting enzyme, and AT1R. These observations indicate that upregulation of TNF-{alpha} during estrogen deficiency may contribute to enhance vascular constriction by altering the vascular angiotensin II system.


Key Words: tumor necrosis factor • aging • estrogen • vasoconstriction • angiotensin II


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMethods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Cardiovascular disease is more prevalent in postmenopausal women compared with premenopausal women. Although epidemiological and experimental evidence indicate that estrogen deficiency triggers some of the alterations in vascular function associated with menopause, the mechanisms are still unclear.

The renin–angiotensin system (RAS) is involved in the regulation of vascular homeostasis. Alterations in the RAS, such as upregulation of angiotensin-converting enzyme (ACE)1 and angiotensin (Ang) II type 1 receptor (AT1R),2–4 have been described to occur in postmenopausal women and ovariectomized (OVX) animals. Hence, it has been hypothesized that Ang II is involved in the alterations of vascular function in postmenopausal women.5,6 Ang II, which is the main effector of the system, can be locally formed in vascular tissues by the vascular RAS. Indeed, endogenous Ang II plays an important role in the control of vascular tone.7,8 For instance, Ang II enhances adrenergic vasoconstriction through activation of AT1R.9,10 An increase in the actions of Ang II via AT1R have been etiologically associated with vascular disease.11

Tumor necrosis factor (TNF)-{alpha} is a proinflammatory cytokine involved in the pathogenesis of some vascular disorders.12,13 In previous studies, we have found that estrogen deficiency is associated with an increase in circulating levels of TNF-{alpha}, which results in vascular dysfunction.14 Interestingly, some of the vascular effects of TNF-{alpha}, such as increased free-radical production, inflammation, and enhanced remodeling, resemble those attributed to Ang II. In fact, both factors share signal transduction pathways, such as activation of mitogen-activated protein kinases15,16 and nuclear factor {kappa}B,17 and we found recently that in endothelial cells TNF-{alpha} mediates Ang II–induced matrix metalloproteinase 2 release.18 Furthermore, in other cell types, TNF-{alpha} has been shown to increase the formation of angiotensinogen,19 as well as the expression of AT1R.20 Thus, we hypothesized that in the state of estrogen deficiency, TNF-{alpha} is a mediator inducing alterations in tissue RAS and promoting Ang II modulation of vasoconstriction.


*    Methods
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 up arrowAbstract
 up arrowIntroduction
 *Methods
down arrowResults
 down arrowDiscussion
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Animal Model
This study was approved by the University of Alberta Health Sciences Animal Policy and Welfare Committee and was in accordance with the Canadian Council on Animal Care and National Institutes of Health guidelines. Female Sprague–Dawley rats were obtained from Charles River (Montreal, Quebec, Canada) and were housed in the facilities of the University of Alberta until experimentation at 12 to 15 months of age. This age was chosen because the animals are approaching a state of reproductive senescence (ie, similar to the postmenopausal state of women); however, rats experience constant estrus at the end of their reproductive age and continue to produce variable levels of estrogen. Thus, to decrease the variability on estrogen levels in these aged animals, we remove the ovaries at the time of the initial treatment and randomly assigned the animals to different treatments for 4 weeks. We have described this model previously.14,21–25

Experimental Design
To investigate the effects of estrogen deficiency on vascular function, OVX rats were treated with either a placebo pellet (n=8) or an estrogen pellet (1.5 mg/pellet, 60 day release, Innovative Research of America; n=8), which results in maximal serum estrogen levels (&80 pg/mL) similar to that of intact cycling rats. Cycling rats (&4 months old; in proestrus; n=6) were used as a reference group. To evaluate the role of elevated TNF-{alpha} that is observed in estrogen deficiency, 2 additional groups of OVX rats were treated with either etanercept ([Etan] a TNF-{alpha} inhibitor, Immunex Corporation), administered SC at 0.3 mg/kg, 3 times a week (n=8), or placebo (SC injection of double distilled H2O; OVX, n=8) for 4 weeks before experimentation. Because results from control animals for estrogen and etanercept treated animals yield similar results and were combined for the final analysis.

Etan is composed of the extracellular ligand–binding portion of the human 75-kDa (p75) TNF-{alpha} receptor 2. Thus, Etan binds and inactivates circulating TNF-{alpha}. The Etan dose for chronic studies was chosen based on effective TNF-{alpha} inhibition from previous studies in humans and rats.14,26 Rats were euthanized by exsanguination while under anesthesia (sodium pentobarbital, &60 mg/kg body weight). A blood sample was taken, and serum was obtained by centrifugation. Serum bioactive TNF-{alpha} was measured using the L929-8 bioassay as described previously.20

Vessel Preparation
A portion of the mesentery was excised and immersed in ice-cold HEPES-buffered physiological saline solution, which contained the following (in mmol/L): NaCl 142, KCl 4.7, MgSO4 1.17, Ca2Cl 1.56, KH2PO4 1.18, HEPES 10, and glucose 5.5. Resistance-sized arteries (diameter: &200 µm) were dissected and connected to an isometric myograph system (Kent Scientific Corp) as described previously.21 Four separate baths were used to study arterial segments simultaneously. Force production was recorded on a data acquisition system (Workbench, Strawberry Tree Inc).

Vascular Function Studies
Sensitivity of mesenteric arteries to adrenergic vasoconstriction was evaluated with phenylephrine (PE). Cumulative concentrations of PE (0.1 to 50 µmol/L) were added to the bath, and force was measured. After completion of each dose–response curve, a 30-minute recovery period was allowed, during which the baths were changed every 10 minutes with fresh HEPES-buffered physiological saline solution.

To investigate the modulation of adrenergic constriction by endogenously produced Ang II, PE constriction curves were generated in the absence or presence of candesartan (10 µmol/L, gift from AstraZeneca, Sweden), a specific AT1R blocker, for 15 minutes before PE concentration–response curves. The concentration of AT1R blocker was calculated based on previous studies.27 Moreover, in a subset of vessels preincubated with different concentrations of candesartan (0.001 to 10 µmol/L), maximal effect on PE vasoconstriction was seen at 1 and 10 µmol/L. Because exogenous Ang II induces only a transient constriction in mesenteric arteries,28 it was not possible to construct concentration–response curves of the direct vasoconstrictor effect of Ang II.

The role of endothelium on PE responses and on Ang II modulation of constriction was evaluated by mechanical removal of the endothelium threading a human hair through the lumen of the artery. Confirmation of complete endothelium removal was assessed pharmacologically with a bolus dose of 1 µmol/L of methacholine. All of the constriction curves were normalized to 100% for individual vessels.

Western Blot Analysis for AT1R and ACE
AT1R and ACE protein expression in mesenteric arteries was evaluated by Western immunoblot. Mesenteric arteries were dissected and homogenized in Eppendorf tubes (containing a protease inhibitor mixture to inhibit serine, cysteine, and aspartic proteases to prevent degradation; Sigma) using a tissue homogenizer. The Bradford assay was used to measure protein concentration. Twenty micrograms of protein were loaded onto an SDS-PAGE 9% gel and transferred to a nitrocellulose membrane. Membranes were then probed with rabbit polyclonal anti-AT1R antibody (1:400; Santa Cruz Biotechnology) or anti-ACE antibody (1:200; Santa Cruz Biotechnology). Specificity of primary antibodies was tested using a specific blocking peptide (Santa Cruz Biotechnology). Primary antibody was preabsorbed for 30 minutes with a 5-times-higher concentration of specific blocking peptide before probing the membranes. The primary antibody was then detected with a peroxidase-conjugated host-specific secondary antibody (1:2000; Santa Cruz Biotechnology). Membranes were scanned with a Fluor Multimager, and bands were quantified by densitometric analysis. After initial exposure to these antibodies, membranes were washed 3 times with 0.1% tween 20 in PBS and then probed with anti {alpha}-tubulin (as a loading control; 1:1000; Santa Cruz Biotechnology).

Immunofluorescence for Ang II Expression on Mesenteric Arteries
Mesenteric arteries were placed in embedding medium (Tissue-Tek, Sakura Finetek USA, Inc) and frozen in liquid nitrogen. Arteries were sectioned (10 µm) using a cryostat and fixed with cold acetone. Slides were then incubated with and without a primary antibody against Ang II (1:100; Peninsula Laboratories) and then exposed to a fluorescent secondary antibody (1:200; Alexafluor 488; Molecular Probes). The Vectashield H-1200 Mounting Kit (Vector Laboratories) was used, and slides were analyzed under a fluorescence microscope (Olympus).

Data Analysis
Data from each dose–response curve was fitted to the Hill equation and a straight line generated by linear least-squares regression analysis. The EC50 for each individual artery was determined from this line and the mean±SE calculated from the curves. Tension (T) was calculated using the formula: T=Force (milliNewtons [mN])/2xaxial length (mm2). ANOVA was used for statistical analysis among groups. Post hoc analysis was performed using Tukey’s test. A Student’s t test was used to compare EC50 between 2 groups. Tests were considered significant at P<0.05.


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 *Results
down arrowDiscussion
 down arrowReferences
 
Estrogen-deficient rats had higher serum levels of TNF-{alpha} compared with estrogen-replaced animals (29.8±4 and 6.9±5 pg/mL, respectively; P<0.05). Treatment with Etan reduced TNF-{alpha} levels in estrogen-deficient animals to levels similar to that of estrogen-replaced rats (10.6±4 pg/mL).

Effects of TNF-{alpha} Inhibition on PE Vasoconstriction
TNF-{alpha} inhibition with Etan decreased the sensitivity to PE vasoconstriction in estrogen-deficient animals (OVX versus Etan: EC50=2.1±0.9 versus 4.6±0.4 µmol/L) to similar levels of that of estrogen-replaced (EC50=3.9±0.4 µmol/L; Figure 1) or cycling rats (EC50=4.7±0.7 µmol/L). There were no differences in maximum tension among groups (OVX, estrogen, and Etan: 3.9±0.7, 3.8±0.5, and 3.9±0.8 mN/mm2, respectively).


Figure 1
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  		Figure 1. Phenylephrine concentration response curves (A) and EC50 bar graphs (B) of mesenteric arteries from OVX rats treated with placebo (OVX; n=8), estrogen replacement (estrogen; n=8), or Etan (n=8). Bars, mean±SE. Different letters indicate values that are statistically significant among groups (P<0.05).

Effects of AT1R Blockade on PE Vasoconstriction
In vitro AT1R blockade with candesartan (10 µmol/L) decreased the sensitivity to PE constriction of vessels from estrogen-deficient rats (EC50=4.40±0.6 µmol/L; Figure 2A) to levels similar to that of estrogen-replaced (EC50=3.9±0.4 µmol/L; Figure 1) or Etan-treated (4.6±0.4 µmol/L; Figure 1) animals. However, candesartan did not significantly alter PE constriction in cycling animals (EC50=4.1±0.2 µmol/L) or in estrogen-depleted animals treated either with estrogen replacement (EC50=2.8±0.4 µmol/L; Figure 2B) or Etan (EC50=4.27±0.3 µmol/L; Figure 2C). Candesartan did not alter maximum tension in OVX-, estrogen-, or Etan-treated animals (3.5±0.8, 4.0±0.7, and 4.21± 0.9 mN/mm2, respectively).


Figure 2
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  		Figure 2. Effects of AT1R blockade with candesartan on PE constrictor responses of mesenteric arteries. PE concentration response curves and EC50 bar graphs in the absence ({circ}) or presence (•) of candesartan (Cand; 10 µmol/L) of mesenteric arteries from OVX rats treated with placebo (OVX, n=8; A), estrogen (E2, n=8; B), or Etan (n=8; C). Bars, mean±SE. *P<0.05 vs PE alone.

Effects of Endothelial Removal on Candesartan Modulation of PE Vasoconstriction in Estrogen-Deficient Animals
Because the endothelium could be involved in the generation of Ang II, we next tested the effects of endothelial denudation on AT1R modulation of vasoconstriction. Endothelial removal did not significantly modify the effects of candesartan (EC50; with versus without endothelium: 4.40±0.6 µmol/L versus 4.51±0.7 µmol/L; Figure 3), indicating that the presence of an intact endothelium is not necessary for the effect of AT1R blockade on modulation of PE constriction.


Figure 3
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  		Figure 3. Effects of endothelial removal on AT1R modulation of PE vasoconstriction in estrogen-deficient animals. PE concentration response curves (A) and EC50 bar graphs (B) of mesenteric arteries from OVX rats treated with placebo in the presence of endothelium (OVX; n=8), with endothelium plus candesartan (OVX+Cand; n=8), and without endothelium plus candesartan (n=4). Bars, mean±SE. Different letters indicate values that are statistically significant among groups (P<0.05).

Effects of TNF-{alpha} Inhibition on AT1R, ACE, and Ang II Expression in Mesenteric Arteries
The expression of AT1R and ACE in mesenteric arteries was evaluated by Western immunoblot. Vascular expression of AT1R and ACE was higher in estrogen-deficient rats compared with either estrogen-replaced rats or TNF-{alpha} inhibition (Figure 4). Ang II expression in mesenteric arteries was evaluated by immunofluorescence. Ang II fluorescent staining was primarily located in the arterial media and was higher in estrogen-depleted animals compared with estrogen- or Etan-treated animals (Figure 5).


Figure 4
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  		Figure 4. ACE and AT1R expression in mesenteric arteries. Representative Western blots and densitometric analysis for ACE (A) and AT1R (B) expression in arteries from placebo (OVX; n=5), estrogen-treated (n=5), or Etan-treated (n=5) animals. Bars, mean±SE. Different letters indicate values that are statistically significant among groups (P<0.05).


Figure 5
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  		Figure 5. Immunofluorescence for Ang II expression in mesenteric arteries from estrogen-deficient rats treated with placebo (OVX), estrogen replacement (Estrogen), or chronic TNF-{alpha} inhibition with Etan (Etan). Images are representative of 4 independent experiments. Vessels were probed with fluorescent antibodies against Ang II (Red). 4',6-Diamidino-2-phenylindole was used for staining of cell nuclei (blue). Autofluorescence (green) shows the arterial elastic internal lamina and vessel wall. B, Densitometric analysis of fluorescence intensity. Bars, mean±SE. Different letters indicate values that are statistically significant among groups (P<0.05).


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
The results of this study demonstrate that treatment with a selective TNF-{alpha} inhibitor in estrogen-deficient rats decreases vascular expression of ACE, Ang II, and AT1R in mesenteric arteries, and these changes were accompanied by a reduction in AT1R modulation of adrenergic vasoconstriction.

TNF-{alpha} is a proinflammatory cytokine involved in the pathogenesis of cardiovascular disease.12,13 Estrogen deficiency is associated with an increase in TNF-{alpha} levels,14,29,30 and we reported previously that upregulation of TNF-{alpha} during estrogen deficiency may result in vascular dysfunction.14,29,30 However, to our knowledge, this is the first study that shows evidence that TNF-{alpha} is a mediator of vascular RAS alterations during estrogen deficiency.

Estrogen deficiency has been associated with an increase in vasoconstriction,21,31–33 which is in part mediated by an increase in the production of endogenous vasoconstrictors.21,31 Ang II has direct vasoconstrictor effects in some vascular beds; however, in other vascular beds, such as small mesenteric arteries, Ang II acts as a facilitator of vasoconstriction. In fact, previous studies have shown that endogenous Ang II mediates adrenergic constriction in different vascular beds,9,10,34–36 including mesenteric arteries,9,10,37 and, accordingly, vasoconstriction to adrenergic agonists, such as PE, may be reduced by Ang II receptor blockers9,10,34,38 or ACE inhibitors.27,35,37 However, whether endogenous Ang II increases vascular tone during the state of estrogen deficiency was unknown.

In the present studies, preincubation of isolated mesenteric arteries from estrogen-deficient animals with a selective AT1R blocker (candesartan) decreased the sensitivity to PE constriction to levels similar to that of estrogen-replaced animals but did not have any significant effect in vessels from animals treated with Etan. These findings suggest that the AT1R-sensitive component of PE vasoconstriction was increased during the state of estrogen deficiency and reduced by TNF-{alpha} inhibition.

AT1R mediates some of the detrimental effects on vascular function attributed to Ang II, such as oxidative stress and inflammation, as well as vasoconstriction.39 We observed that estrogen-deficient animals had a &2-fold increase in AT1R protein expression in mesenteric arteries, which was reduced by treatment with Etan. Interestingly, the reduction in AT1R by TNF-{alpha} antagonism was associated with a decrease in candesartan modulation of PE constriction, suggesting that a decrease in AT1R expression in estrogen-depleted animals by Etan may in part mediate the decrease in vasoconstriction seen in this group.

Estrogens have direct inhibitory effects on AT1R expression40; thus, both the lack of estrogens, and the increase in TNF-{alpha} levels may have contributed to upregulate vascular AT1R in this model. It is unclear, however, if the upregulation of AT1R expression during estrogen deficiency requires the interplay of TNF-{alpha} with other factors present in the state of estrogen deficiency. For instance, we have shown previously that the increase in TNF-{alpha} levels with estrogen deficiency decreases the availability of NO,14 which has negative effects on AT1R expression.41 However, in isolated rat vascular smooth muscle cells (data not shown), as well as in other cell types,42 TNF-{alpha} solely was able to upregulate AT1R expression.

Alterations in tissue Ang II levels may also result in changes on AT1R tissue expression. Ang can be sequestered from the circulation or generated in vascular tissues, because all of the components for Ang II formation (renin, angiotensinogen, and ACE) can be found in the vascular wall.43 Indeed, Ang II formation has been demonstrated to occur in endothelial cells,44 as well as in vascular smooth muscle cells.45,46 We observed that Ang II expression was increased in mesenteric arteries from estrogen-depleted animals compared with estrogen-replaced animals. Strikingly, TNF-{alpha} inhibition with Etan was also associated with a reduction in Ang II expression. Whether Ang II was taken up from circulating plasma and/or whether it was generated in the vascular tissue cannot be answered for certain from our observations. However, studies have shown that the level of ACE expression in vascular tissues correlates with the rate of local Ang II formation.7,47 Accordingly, we found that estrogen-deficient animals have an &3 fold-increase in vascular ACE expression compared with estrogen-replaced animals, which was reduced to levels similar to that of estrogen-deficient animals with the TNF-{alpha} inhibitor. These results agree with previous studies reporting that estrogen deficiency is associated with an increase in ACE expression1 and suggest that TNF-{alpha} may directly or indirectly participate in the regulation of ACE expression. Contrary to these findings, it has been reported that TNF-{alpha} downregulates ACE expression in human umbilical endothelial cells.48 It is important to point out that the protein for tissue analysis in our experiments was obtained from homogenates of complete mesenteric arteries. Additionally, we did not see differences in the expression of ACE across vascular layers of mesenteric arteries using immunofluorescence (data not shown). Thus, it is likely that regional differences in endothelial cell function, as well as differences in experimental conditions, such as concentration of TNF-{alpha}, time of exposure, and the presence of other factors in vivo versus in vitro, may account for this apparent discrepancy.

It is intriguing that higher levels of Ang II were associated with higher expression of AT1R in vessels from aged animals, since Ang II is known to downregulate the expression of this receptor. Interestingly, similar results were seen in endothelial cells in vitro, in which Ang II was unable to downregulate its own receptor.40 We investigated whether the endothelium could be the primary source of endogenous Ang II in estrogen-deficient animals. However, endothelial removal did not alter the effects of AT1R blockade on vasoconstriction suggesting that the endothelium is not necessary for vascular Ang II formation. In agreement with these observations, Leite et al49 found that endothelium-denuded mesenteric arteries were still able to produce Ang II.

Our results suggest that vascular RAS contributes to enhance adrenergic vasoconstriction in mesenteric arteries from estrogen-deficient animals and that this mechanism was attenuated by TNF-{alpha} inhibition. In a recent study,50 Wassmann et al50 found that AT1R antagonism improved flow-mediated vasodilation in postmenopausal women who were not taking hormone replacement therapy, but it was not effective in women treated with hormone replacement therapy, suggesting that the lack of ovarian hormones facilitates the detrimental effects of Ang II. Thus, an increase in TNF-{alpha} during estrogen deficiency may result in upregulation of AT1R in resistance arteries, which may contribute to increased vasoconstriction in the state of estrogen deficiency (eg, postmenopausal women).

In our previous studies we have shown that TNF-{alpha} inhibition improves NO modulation of vascular function in a model of estrogen deficiency,14 and some studies have also shown that NO and Ang II interact in the modulation of vascular function.41,51 In fact, NO modulates AT1R expression41 and can downmodulate the vasoconstriction to Ang II.51 Thus, an increase in NO bioavailability by TNF-{alpha} inhibition could have contributed to reduce AT1R expression resulting in decreased Ang II–evoked vasoconstriction. On the other hand, Ang II is known to increase oxidative stress by stimulating the generation of superoxide anion from reduced nicotinamide-adenine dinucleotide phosphate oxidase, which can inactivate NO. Thus, a decrease in the effect of Ang II via AT1R caused by the treatment with Etan may contribute to decrease oxidative stress and NO inactivation, resulting in higher NO availability.

Perspectives
All together our findings support the hypothesis that TNF-{alpha} is a mediator of vascular dysfunction during estrogen deficiency and that an increase in TNF-{alpha} levels may result in alterations of vascular RAS. One consequence of these changes is an increase in adrenergic constriction, which has been described to occur in postmenopausal women,33,52 and has been implicated in the pathogenesis of vascular disorders, such as hypertension.53,54 Importantly, Ang II and TNF-{alpha} share similar signaling pathways,15–17 and both factors can induce similar responses in vascular cells. Hence, these factors could initiate a positive feedback loop that may potentiate the actions of each other in the vascular wall.


*    Acknowledgments
 
Sources of Funding

The Canadian Institute for Health Research supported this study. S.T.D. is a Canada Research Chair in Women’s Cardiovascular Health and an Alberta Heritage Foundation for Medical Research Scientist. I.A.A. is a fellow of Alberta Heritage Foundation for Medical Research and Tomorrow’s Research Cardiovascular Health Professionals. S.T.D. received less than $10 000 in other research support.

Disclosures

None.

Received February 10, 2006; first decision February 28, 2006; accepted March 17, 2006.


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 *References
 

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