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Am J Physiol Heart Circ Physiol 290: H30-H36, 2006. First published September 2, 2005; doi:10.1152/ajpheart.00349.2005
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Cyclooxygenase-2 induction by bradykinin in aortic vascular smooth muscle cells

Jorge A. Rodriguez, Paula De la Cerda, Eileen Collyer, Valerie Decap, Carlos P. Vio, and Victoria Velarde

Departamento de Fisiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile

Submitted 8 April 2005 ; accepted in final form 31 August 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vascular smooth muscle cell proliferation and migration play an important role in the pathophysiology of several vascular diseases, including atherosclerosis. Prostaglandins that have been implicated in this process are synthesized by two isoforms of cyclooxygenase (COX), with the expression of the regulated COX-2 isoform increased in atherosclerotic plaques. Bradykinin (BK), a vasoactive peptide increased in inflammation, induces the formation of prostaglandins through specific receptor activation. We hypothesized that BK plays an important role in the regulation of COX-2, contributing to the increase in production of prostaglandins in vascular smooth muscle cells. Herein we examined the signaling pathways that participate in the BK regulation of COX-2 protein levels in primary cultured aortic vascular smooth muscle cells. We observed an increase in COX-2 protein levels induced by BK that was maximal at 24 h. This increase was blocked by a B2 kinin receptor antagonist but not a B1 receptor antagonist, suggesting that the B2 receptor is involved in this pathway. In addition, we conclude that the activation of mitogen-activated protein kinases p42/p44, protein kinase C, and nitric oxide synthase is necessary for the increase in COX-2 levels induced by BK because either of the specific inhibitors for these enzymes blocked the effect of BK. Using a similar approach, we further demonstrated that reactive oxygen species and cAMP were not mediators on this pathway. These results suggest that BK activates several intracellular pathways that act in combination to increase COX-2 protein levels. This study suggests a role for BK on the evolution of the atheromatous plaque by virtue of controlling the levels of COX-2.

atherosclerosis; signal transduction; antioxidants

PROSTAGLANDINS such as prostacyclin (PGI2), thromboxane A2 (TxA2), and prostaglandin E2 (PGE2), are widely recognized as inflammatory mediators that also regulate vascular contractility. Prostaglandins are produced by the conversion of membrane-derived arachidonic acid (AA) through the enzymatic activity of cyclooxygenase (COX). COX is a rate-limiting enzyme and exists in two distinct isoforms: COX-1, which is constitutively expressed, and COX-2, which can be induced by shear stress and many growth factors. This inducible isoform is expressed at sites of inflammation such as atherosclerotic lesions from native and transplanted human coronary arteries (24), where it is found in macrophages, endothelial, and smooth muscle cells (1).

Proliferation and migration of vascular smooth muscle cells (VSMC) in arteries play an important role in the pathophysiology of atherosclerosis, hypertension, and restenosis after angioplasty (11). On activation, VSMC proliferate, migrate into the subendothelium, and secrete extracellular matrix proteins, stabilizing the plaque and reducing the risk of complications.

Bradykinin (BK) is a peptide hormone that is formed locally in body tissues and fluids from its plasma precursor kininogen during physiological and inflammatory processes. BK elicits several actions such as vasodilation, increased vascular permeability, and recruitment of inflammatory cells to the site of injury. All these events occur via two BK receptor subtypes, the B2 receptor (B2KR), which is constitutively expressed in VSMC and endothelial cells, and the B1 receptor (B1KR), which is induced by inflammation and injury. In endothelial cells, BK induces the activation of the endothelial nitric oxide (NO) synthase (eNOS) with the production of NO along with the activation of phospholipase A2, the release of AA, and the production of prostaglandins. NO and prostaglandins act on the smooth muscle (10, 23). In contrast, when BK interacts directly with its receptors in VSMC, it induces the activation of several enzymes, such as mitogen-activated protein kinases (MAPKs) and protein kinase C (PKC), along with causing the generation of reactive oxygen species (ROS), cellular migration, and the production of extracellular matrix proteins (9, 26, 27).

Several studies have reported the ability of BK to regulate COX-2 expression in different cell types. In endothelial cells in culture, BK induces COX-2 mRNA expression after 4 h of incubation (4). In addition, in smooth muscle cells from human airways, it has been shown that BK stimulates COX-2 activity and expression in a time- and dose-dependent manner and that this regulation is implicated in asthma (3, 18).

Recent studies using COX-2 inhibitors have suggested that this enzyme has both pro- and antiatherogenic effects (16). This apparent contradiction can be understood by considering that certain tissues express more of one isoform than the other (platelets express mainly COX-1, whereas vascular tissue expresses mainly COX-2). In addition, depending on the localization, each isoform synthesizes preferably one product over others (TxA2 in platelets vs. PGI2 in VSMC). Another important consideration is the time frame of COX-2 expression (initial/acute or late/chronic) within the atheromatous plaque. The regulation of COX-2 function on VSMC from vessels subjected to high pressure has not been fully studied, and its elucidation will help to understand the role of this enzyme in atheromatosis. Considering the relevant participation of BK on the production of prostaglandins in vascular function and its potential role in the development of the atherosclerotic plaque, we hypothesize that BK participates in the regulation of COX-2 expression in aortic VSMC.

In this study we examined the modulation of COX-2 protein levels by BK in primary cultured VSMC. In addition, we further elucidated the signaling pathways involved in this process, including the contribution of ROS as signaling mediators.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
VSMC culture. This investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996) and were approved by the Institutional Bioethics Committee in compliance with the Guiding Principles in the Care and Use of Animals endorsed by the American Physiological Society and certified by the National Fund of Science and Technology from Chile.

Aortic VSMC from 75- to 150-g male Sprague-Dawley rats were prepared by a modification of the method of Majack and Clowes (15). Explants were cultured in DMEM (GIBCO-BRL, Gaithersburg, MD) with antibiotics and 10% fetal calf serum at 37°C in a humidified atmosphere. Cells were identified as VSMC by their characteristic morphology and by their positive staining against smooth muscle cell {alpha}-actin and negative staining against Von-Willebrand's factor antibodies. Cells in culture acquired a proliferative state with the typical "hill and valley" growth pattern. Cells between passages 2 and 6 were used in all experiments. Quiescence was induced by culturing cells in DMEM without serum for 24 h before experimentation (26).

To determine the effect of BK on COX-2 levels, VSMC were cultured in the absence of serum and the presence of BK for different time points. At the end of the incubation period, cells were harvested, and proteins were used for the different determinations. For the studies on the pathways involved in the effect of BK, cells were preincubated for 30–120 min with the inhibitors or the antagonists previous to the 24-h stimulation with BK. Antagonists were maintained in the medium for the whole incubation period.

Immunohistochemistry. Slices from aortas were immunostained according to the peroxidase-antiperoxidase (PAP) method (25), with modifications for COX-2 staining (28). Briefly, sections were incubated with primary anti-COX-2 antibody (1:200, SC-1747, Santa Cruz, CA) overnight at 22°C, followed by the secondary antibody (1:20) and PAP complex (1:150). Peroxidase activity was visualized with 3,3'-diaminobenzidine and hydrogen peroxidase. Sections were counterstained with hematoxylin.

VSMC cultured in coverslips were fixed in 4% paraformaldehyde-0.1% glutaraldehyde, dehydrated, and stained for COX-2 using the same procedure as for tissue slices. Sections and cells were observed and photographed on a Nikon Eclipse 600 microscope with a Nikon DXM1200 digital photographic system (Nikon, Tokyo, Japan).

Western blotting. Proteins (20 µg/well) were separated by 12% SDS-PAGE and transferred to 0.45-µm polyvinylidene difluoride membranes. Membranes were blotted overnight at 4°C with a polyclonal anti-COX-2 antibody (1:2,000, Cayman Chemicals, Ann Arbor, MI), stripped, and reblotted with a polyclonal anti-tubulin antibody (1:4,000, Sigma) that acted as a loading control. Immunoreactive bands were visualized by a chemiluminescent method (Western Lightning, Perkin Elmer) and Kodak X-LS film. Densitometric analysis was performed by using the public domain NIH Image program v1.61 (developed at the National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image) as previously described (26).

Statistical analysis. Data are expressed as means ± SE and were analyzed by the nonparametric test of Kruskal-Wallis for unpaired samples. Values were considered significant when P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
COX-2 protein localization in rat aorta and primary cultures of VSMC. Under basal conditions, we observed COX-2 immunostaining in the endothelium, in sporadic cells of the smooth muscle layer, and in the vasa vasorum of the adventitia (Fig. 1, A–C). We also observed COX-2 immunostaining in sporadic cells from primary cultures of VSMC under basal conditions (Fig. 1D). In addition, when stimulated with BK (10–7 M) for 24 h, the number of COX-2-immunoreactive cells was increased (Fig. 1E).



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  		Fig. 1. Cyclooxygenase-2 (COX-2) protein localization in rat aorta and primary cultures of VSMC. Aortas from normal rats (A–C) and primary cultures of vascular smooth muscle cells (VSMC) from rat aortas (D and E) were immunostained using an anti-COX-2 antibody. No staining was observed when a nonimmune antibody was used as a control (not shown). Micrographs show staining in endothelial cells (arrowhead), smooth muscle cells (arrows), and in vasa vasorum (*) (A). A higher magnification of the inmunostaining in vasa vasorum is shown in B and in some smooth muscle cells is shown in C. VSMC from control cultures are shown in D and stimulated with bradykinin (BK) for 24 h are shown in E. Bars, 50 µm.

 
Effects of BK on COX-2 protein levels. A concentration-response curve for BK was performed with concentrations from 10–10 to 10–6 M BK, reaching a plateau at 10–7 M (data not shown); we used this concentration for the following experiments. BK at 10–7 M caused a significant increase in COX-2 protein levels (Fig. 2), reaching maximal levels at 24 h (longest observation period); at this time point, COX-2 levels were 2.6 ± 0.6-fold higher relative to control. At 8 h a slight but nonsignificant decrease in COX-2 levels was observed.



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  		Fig. 2. Effects of BK on COX-2 protein levels. VSMC were incubated with BK (0.1 µM) for stated time periods. Bars represent means ± SE for 4 experiments. *P < 0.05 vs. control. Bottom: representative Western blot. OD, optical density.

 
Participation of ROS in BK-induced COX-2 protein levels. We have previously observed that ROS are produced by BK-activated signaling pathways (9). To assess the role of ROS in the BK-activated signaling cascade that increases the levels of COX-2 protein, we preincubated VSMC with one of the following antioxidants: diphenylene iodonium (DPI, 10 µM, 30 min) or lipoic acid (LA; 100 µM, 120 min). The preincubation was followed by application of BK (0.1 µM) for 24 h. As shown in Fig. 3, the BK-mediated increase in COX-2 protein levels (2.42 ± 0.45-fold of control) was not modified by DPI (2.06 ± 0.27-fold of control) or LA (3.08 ± 0.78-fold of control).



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  		Fig. 3. BK increases COX-2 protein through a pathway that is not mediated by reactive oxygen species (ROS). VSMC were preincubated with the antioxidants diphenylene iodonium (DPI; 10 µM, 30 min) or lipoic acid (LA; 100 µM, 120 min) followed by the incubation with BK (0.1 µM) for 24 h. Bars represent means ± SE for 4 experiments. *P < 0.05 vs. control. Bottom: representative Western blot.

 
Participation of MAPK in BK-induced COX-2 protein levels. MAPKs are activated by BK (26). To determine whether one or more enzymes of this family were involved in the signaling pathway, we preincubated VSMC for 30 min, with either the MEK1/2 inhibitor UO126 (1 µM) or the p38 MAPK inhibitor SB-203580 (1 µM) followed by the incubation with BK (0.1 µM) for 24 h. UO126 significantly blocked COX-2 levels in response to BK (3.9 ± 1.6 vs. 1.7 ± 0.5-fold of control, P < 0.05), whereas SB-203580 did not modify them (Fig. 4), suggesting the participation of the MAPK p42/p44 but not of the p38 MAPK pathway.



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  		Fig. 4. BK increases COX-2 protein through a pathway that is mediated by the MAPK p44/p42 but not p38-MAPK. VSMC were preincubated for 30 min with the MEK inhibitor UO126 (1 µM) or the p38-MAPK inhibitor SB-203580 (1 µM) followed by the incubation with BK (0.1 µM) for 24 h. Bars represent means ± SE for 4 experiments. *P < 0.05 vs. control, {dagger}P < 0.05 vs. BK. Bottom: representative Western blot.

 
Participation of cAMP and protein kinase A in BK-induced COX-2 protein levels. In VSMC from pulmonary aorta, the participation of cAMP in COX-2 induction has been reported (3). To determine whether in our model this pathway was involved, we preincubated VSMC for 30 min with either the adenylate cyclase inhibitor 2'-5'-dideoxyadenosine (DDA, 10 µM) or the protein kinase A (PKA) inhibitor H-89 (5 µM) followed by the incubation with BK (0.1 µM) for 24 h. Although DDA inhibited COX-2 expression in response to BK, we did not see an inhibition of COX-2 induction by BK in the presence of H-89; moreover, the preincubation with H-89 in the absence of BK was sufficient stimulus to increase COX-2 levels in VSMC (1.8 ± 0.9-fold of control) (Fig. 5). These results suggest the participation of cAMP production in COX-2 induction in aortic VSMC. The effects of H-89 on COX-2 induction could be due to the actions of this inhibitor in other kinases.



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  		Fig. 5. BK increases COX-2 protein through a pathway that is mediated by cAMP but not protein kinase A (PKA). VSMC were preincubated for 30 min with the adenylate cyclase inhibitor 2'-5'-dideoxyadenosine, (DDA, 10 µM) or the PKA inhibitor H-89 (5 µM) followed by the incubation with BK (0.1 µM) for 24 h. Bars represent means ± SE for 4 experiments. *P < 0.05 vs. control, {dagger}P < 0.05 vs. BK. Bottom: representative Western blot.

 
PKC and NO mediate the BK-activated pathway that induces COX-2 levels. To determine whether PKC and NOS were involved in the pathways activated by BK to increase COX-2 levels, we preincubated VSMC for 30 min with either the PKC inhibitor bisindolylmaleimide I (Bim I, 4 µM) or the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 0.5 mM), followed by incubation with BK (0.1 µM) for 24 h. Both inhibitors blocked COX-2 expression in response to BK (1.24 ± 0.29-fold and 1.62 ± 0.40-fold of control, respectively) compared with BK (3.58 ± 0.65-fold of control) (Fig. 6). To confirm that these cells were producing NO, we measured nitrites and found a twofold increase in these metabolites after the incubation with BK (12.3 ± 0.9 vs. 24.9 ± 3.5 nmol/mg, respectively). This increase was blocked when cells were preincubated with L-NAME. To identify the origin of NO, we also measured the presence of neuronal (nNOS) and inducible NOS (iNOS), observing an intense band for nNOS and a rather faint band for iNOS in these cells (data not shown).



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  		Fig. 6. BK increases COX-2 protein through a pathway mediated by protein kinase C (PKC) and nitric oxide (NO). VSMC were preincubated for 30 min with the PKC inhibitor bisindolylmaleimide I (Bim I; 4 µM) or the NO synthase (NOS) inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 0.5 mM) followed by the incubation with BK (0.1 µM) for 24 h. BK-induced increase in COX-2 protein was blocked by both inhibitors. Bars represent means ± SE for 4 experiments. *P < 0.05 vs. control, {dagger}P < 0.05 vs. BK. Bottom: representative Western blot.

 
BK induces COX-2 protein levels via the B2 receptor. To determine which of the two receptors for BK mediates the signaling that induces COX-2 protein levels, we preincubated VSMC for 30 min with the B2KR specific antagonist HOE-140 (1 µM) or the B1KR specific antagonist [D-Arg9-Leu8]BK (Leu-8, 1 µM) followed by the incubation with BK (0.1 µM) for 24 h. BK-induced increase in COX-2 protein was blocked by HOE-140. Leu-8 did not significantly inhibit BK induction on COX-2 (Fig. 7A). To further prove that the response was mediated by the B2KR, we stimulated the cells for 24 h with BK (0.1 µM), which is a B2KR agonist, or [D-Arg9]BK (0.1 µM), which is the B1KR agonist. An increase in COX-2 levels was only observed when the B2KR agonist was used (Fig. 7B), confirming the previous result.



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  		Fig. 7. BK increases COX-2 protein via the B2-kinin receptor. A: VSMC were preincubated for 30 min with the B1-kinin antagonist [D-Arg9-Leu8]BK (Leu-8; 1 µM) or the B2-kinin antagonist HOE-140 (1 µM) followed by the incubation with BK (0.1 µM) for 24 h. B: VSMC were incubated for 24 h with the B1-kinin agonist [D-Arg9]BK (dA-BK; 0.1 µM) or the B2-kinin agonist BK (0.1 µM). Bars represent means ± SE for 4 experiments. *P < 0.05 vs. control, {dagger}P < 0.05 vs. BK. Representative Western blots are shown at bottom of A and B.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
COX-2 has long been considered an inducible enzyme that is not present in normal conditions. The finding of immunoreactivity for COX-2 in a normal artery is consistent with previous observations in normal kidney (29) where sporadic cells showed positive staining in the renal cortex. In the kidney, this expression can be increased by several stimuli such as adrenalectomy (28), extracellular calcium concentrations (30), and also BK (20). Similarly, we have observed that BK can increase COX-2 immunostaining and protein levels in VSMC.

In a previous report (26), we have demonstrated that BK induces the activation of MAPK p42/p44 with a maximum at 5–10 min. This early activation has been considered enough to activate the signaling cascade that follows. In fact, preliminary data have shown that 2 min of BK in the medium is enough to induce MAPK phosphorylation, maintaining the maximum at 5–10 min. In this report, we investigated whether BK activates this same pathway to increase COX-2 protein levels. Our observations that UO126 but not SB-203580 decreases COX-2 levels in response to BK suggest that MAPK p42/p44 but not p38 MAPK are mediating the effects of BK in this process. At this same time frame, MAPK p42/p44 phosphorylation in response to BK was not different from control. Our results differ from observations of other investigators in cells from the thick ascending limb of the loop of Henle (5) or from human pulmonary artery smooth muscle cells (4), where they found that p38 MAPK was mediating COX-2 induction. We speculate that origin of VSMC (pulmonary artery vs. aorta) or cell type could explain these differences.

It has been reported that BK, through its receptors, can activate different G proteins, such as Gq, Gi, or Gs (14); for this reason, we have studied the participation of a pathway mediated by the activation of Gs, the cAMP/PKA pathway, on the induction of COX-2 by BK on aortic smooth muscle cells. In agreement with reports by Bradbury et al. (3) in pulmonary artery smooth muscle cells, our results using the adenylate cyclase inhibitor DDA suggested the participation of cAMP in the pathway that leads to COX-2 expression. Surprisingly H-89, a PKA inhibitor, did not block BK-induced COX-2 levels; in fact cells expressed more COX-2 when this enzyme was blocked, even in the absence of BK. The results make us speculate that at the concentrations used in our experiments H-89 could be inhibiting other kinases in addition to PKA, such as MSK1 or S6K1 (8), and that these inhibitions could be producing a more complex regulatory loop.

With regard to the pathway activated by Gq, we studied the effect of PKC and NOS inhibition on COX-2 levels. In this case, the inhibition of the increase in COX-2 levels by BK in the presence of Bim I (a PKC inhibitor) and L-NAME (a NOS inhibitor) suggested the participation of these enzymes in the pathway leading to COX-2 production. Although we have previously demonstrated the presence of PKC-{beta}1 and -{beta}2, -{epsilon}, and -{zeta} in VSMC (6) and that PKC-{epsilon} has been suggested as the mediator for the BK effect on COX-2 expression in airway smooth muscle cells (19), the determination of which of these isoforms is mediating the expression of COX-2 was beyond the aim of this report. In relation to NOS, NO has been involved as a signaling molecule in the pathway leading to COX-2 induction in bone cells (12, 17). In VSMC, NO can be synthesized by the neuronal form of NOS, which has been described to be present in normal conditions in this cell type (22) or by the inducible form of NOS, which is rapidly stimulated under inflammatory conditions (21); according to our results both isoforms could be contributing to the production of NO.

The inducible B1KR is expressed in pathological conditions, whereas the B2KR is expressed constitutively and is believed to participate in the physiological functions of the vessel wall. The stimulatory effect of BK on COX-2 levels was inhibited by the B2KR antagonist but not by the B1KR antagonist. These observations suggest that the B2KR mediates BK effects in VSMC and are in agreement with a recent report that showed that the B2 receptor is necessary for full expression of COX-2 in kidney (13).

Our previous results (27) demonstrated that BK induces an increase in ROS production with a maximum at 30 min. This early stimulus has been considered enough to activate the signaling cascade that follows; for this reason we did not study ROS production at 24 h of stimulation.

Having previously reported that BK induces the production of ROS in these cells (27), we wanted to determine whether ROS were participating in the signaling pathway activated by BK to increase COX-2 levels. Although several studies have reported the participation of ROS on COX-2 expression (2), neither LA, an oxidant scavenger, nor DPI, which has been considered a NADPH oxidase inhibitor, blocked the stimulatory effect of BK on COX-2 levels. These results suggest to us that ROS are not participating on the pathway that leads to increased COX-2 levels. Our study has investigated the regulation of BK on COX-2 levels in smooth muscle cells from aorta, a vessel that is permanently under high pressure and consequently at risk from developing atheromatous plaques. Although the participation of COX-2 in inflammation and other pathological conditions is well known, its pro- or antiatherogenic properties are poorly defined. Macrophage COX-2 has inflammatory and proatherogenic properties, and its derived prostaglandins are related to the instability of the plaque (7). However, COX-2 from endothelial cells is related to prostacyclin synthesis and is antithrombotic and antiatherogenic. Along with macrophages and endothelial cells from the atheroma, COX-2 is also expressed in VSMC. We demonstrated that BK can induce the expression of COX-2 in VSMC and postulate that prostaglandins derived from COX-2 may regulate VSMC proliferation and/or extracellular matrix synthesis, which could result in the stabilization of the plaque and a lower probability of rupture.

This study suggests a role for BK on the evolution of the atheromatous plaque by virtue of controlling the levels of COX-2.

Our observations support the conclusions that 1) BK increases COX-2 protein levels in VSMC; 2) the increase in COX-2 protein is mediated by the B2 kinin receptor, MAPK p42/p44, PKC, and NOS; and 3) ROS are not important regulators of COX-2 levels in this pathway. The role of COX-2 in the atheroma may be cell type specific, and we suggest that the equilibrium between macrophages, endothelium, and VSMC will determine the final effect of COX-2 in the development of the plaque.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Grants Direction of Investigation and Postgraduate from Catholic University (DIPUC 2754–005), Chilean National Fund for Science and Technology (FONDECYT 1040809) (V. Velarde), FONDECYT 1050977 (C. P. Vio), and a doctoral fellowship from Chilean National Committee of Science and Technology (CONICYT) (J. A. Rodriguez).


   ACKNOWLEDGMENTS
 
The expert technical assistance of Maria Alcoholado, Carlos Cespedes, and Marcelo Alonso is gratefully acknowledged.

Present address of J. Rodriguez: Facultad de Ciencias de la Salud, Universidad Andrés Bello, Santiago, Chile.


   FOOTNOTES
 

Address for reprint requests and other correspondence: V. Velarde, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Alameda 340, PO Box 114D, Santiago, Chile (e-mail: mvelarde{at}bio.puc.cl)

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.


   REFERENCES
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 ABSTRACT
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 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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