Journal of the American Society of Nephrology 1683

Heparin Suppresses Endothelin-1 Peptide and mRNAExpression in Cultured Endothelial Cells of SpontaneouslyHypertensive Rats1’2

Koji Yokokawa,3 Masakazu Kohne, Anil K. Mandal, Hideki Tahara, Masashi Yanagisawa, and

Tadano Takeda

K. Yokokawa, M. Kohne, 1. Takeda, First Department

of Internal Medicine, Osaka City University MedicalSchool. Osaka. Japan

H. Tahara. Second Department of Internal Medicine,

Osaka City University Medical School. Osaka. Japan

AK. Mandal, Department of Medicine. Wright StateUniversity at Veterans Affairs Medical Center, Day-ton, OH

M. Yanagisawa, Howard Hughes Medical Institute and

Department of Molecular Genetics. University of Texas

Southwestern Medical Center at Dallas, TX

(J. Am. Soc. Nephrol. 1994; 4:1683-1689)

ABSTRACTHeparin given sc consistently lowers blood pressure

in spontaneously hypertensive rats (SHR). This study

was designed to examine heparin’s effect on vaso-constrictor endothelin- I production in cultured aortic

endothelial cells (EC). Aortic EC from SHR and nor-motensive Wistar Kyoto rats (WKY) were cultured andincubated with or without different concentrations ofheparin. Heparin suppressed endothelin- I release

and endott’ielin-l mRNA expression in a dose- and atime-dependent fashion in both WKY and SHR. Thesuppressive effects were more augmented in SHR

than in WKY: SHR versus WKY-endothelin-1 level at

6 h = 8 ± 1.8 versus 14 ± 2.2 pg/la6 cells (P< 0.01)

and mRNA expression-85 versus 52% maximal in-hibition by heparin, 10 U/mL (=70 �g/mL) (P< 0.01).

When heparin was added with transcriptional inhibi-tor actinomycin D and incubated for 30 mm, no

further inhibition of endothelin-1 mRNA level meas-

I Received January 5, 1993. Accepted August 30, 1993.

2 This work was presented at the annual meeting of American Federation for

Clinical Research, April 30-May 3. 1992, Baltimore, MD, and received the HenryCMstlan Memorial Award.3Correspondence to Dr. AK. Mandal, Department of Medicine, Nephrologyiaboratory, Wright State University at VAMC Campus, 4100 West Third Street,Dayton, OH 45428.

1046-6673/0409-1683503.00/0Journal of the American Society of NephrologyCopyright © 1994 by the American Society of Nephrology

ured after another 30 minutes was observed com-

pared with the endothelin-1 mRNA level in cultured

EC of SHR treated with just actinomycin D at 30 mm.

These results suggest that heparin regulates endog-enous endothelin-1 production by cultured EC, prob-

ably at the transcriptional level, and that this effectis more marked in SHR than in WKY.

Key Words: Hepcirin, endothelin, spontaneously hypertensive

rats, endothelial cell, production

H epanin, a glycosammnoglycan composed of me-peating glucosammne and umonic acid sugar nes-

idues, is a potent inhibitor of vascular smooth muscle

cell (VSMC) proliferation ( 1 ). A number of studieshave been performed to understand the mechanismsby which hepanin inhibits VSMC growth. The anti-proliferative activity may be related to the interaction

of hepanln with hepamin-bmnding growth factors,

namely, fibmoblast growth factors (2), or may be me-hated to its ability to bind other growth factors and

matrix molecules (3). Heparln does not act merely bybinding serum components, because VSMC can bestimulated to proliferate by serum depleted of hepa-

mm-binding growth factors, but it can inhibit thegrowth of VSMC in response to the serum (4). Hepanmndirectly suppresses the induction of c-fos and c-myc

mRNA in VSMC of rat and calf ( 1 ), which may be onemechanism by which hepanln inhibits VSMC pnohlf-emation. Several investigators and we have reportedthat hepanin given sc lowers blood pressure signifi-

cantly in various hypertensive animal models (5). Themechanism of this antihypertensive effect of heparinis not yet fully elucidated; however, the inhibition ofVSMC proliferation appears to be a prime target.

Endothelin- 1 , which has recently been isolated fromthe supemnatant of cultured endothehiah cells (EC) (6),exerts a potent vasoconstrictom effect on a variety of

blood vessels, such as renal artery (7), and producesa sustained elevation of systemic blood pressure in

anesthetized dogs (8) and rats (7). Several studieshave demonstrated that plasma endothelin- 1 levelswere significantly elevated in hypertensive patients

(9, 1 0). We have also shown that an elevated level of

plasma endothelln-1 induces high blood pressure inpatients with endothelin-secreting malignant he-

Heparin. Inhibition of Endothelin Production

1684 Volume 4 ‘ Number 9 ‘ 1994

mangioendothehioma ( 1 1 ), suggesting that endothe-hin- 1 is involved In the development or maintenanceof hypertension (9, 12). Endothebin-1 also stimulatesc-fos and c-myc mRNA expression and the prohifem-ation of VSMC ( 1 3, 1 4). Recently, we have shown thatheparin suppresses endothehin- 1 action and produc-tion in spontaneously hypertensive rats (SHR). mdi-

dating that the antihypertensive effect of hepanmn isat least, in part mediated by the inhibition of endo-thelin- 1 production by EC and by its action on VSMC

(5).This study was designed to examine whether hep-

arm affects endothebin- 1 mRNA expression and tofurther elucidate the inhibitory effect of hepamin onendothehin- 1 production.

MATERIALS AND METHODS

Spontaneously hypertensive and normotensiveWistar Kyoto (WKY) rats were obtained from JapanSLC Inc. (Shizuoka, Japan). Synthetic endothehin-1was obtained from Peptide Institute Co. (Tokyo, Ja-

pan). Dulbecco’s modified Eagle medium (DMEM),penicillin, streptomydin, and fetal calf serum were

obtained from Gibco Laboratories (Grand Island, NY).EC growth supplement (ECGS), hepanin, angiotensinII, and 1 2-O-tetradecanoylphorbol- 13-acetate (TPA)were purchased from Sigma Chemical Co. (St. Louis,MO). Endothebin-1 antibody was obtained from Pen-

insula Lab. Inc. (Belmont, CA). ‘25I-babeled endothe-bin-i, (a-32P]dCTP, and multipnime DNA labeling sys-tem were obtained from Amersham Co. (Tokyo,

Japan).

EC Culture

EC were obtained from the aorta in 1 2-wk-old WKYand SHR and were cultured by the explant method

described previously (5). The cells were cultured instandard culture flasks and incubated at 37#{176}Cwith

atmospheric air and 5% CO2 in DMEM containing 5%fetal calf serum, penicillin (50 �U/mL), streptomycin(50 �tg/mL), and ECGS (50 �tg/mL). EC purity wasassessed by phase contrast microscopy with chanac-teristic “cobblestone appearance” and by immunoflu-

omescence staining with a Factor VIII antiserum.VSMC contamination was less than 0. 1 % . The cul-

tured EC had four to six passages before being usedin this study.

For the measurement of the immunoreactive en-

dothelin- 1 level and the expression of endothebin- 1mRNA, cells were cultured on six-well plates and

standard cultured flasks. respectively. When cellswere confluent, the medium was replaced and incu-

bated with fresh DMEM without serum and ECGS for18 h. After that, the culture medium was removedagain and replaced with serum-free DMEM withoutECGS. EC were incubated without hepanin or with

various concentration of hepamin. After incubation,

the medium in the six-well plates was aspirated,mixed with EDTA, and stored at -80#{176}Cuntil assayed.On the other hand, the cells in flasks after incubation

were used for RNA isolation.

Extraction and RIA of Endothelin-1

Endothelin- 1 was extracted from 4-mL culture me-

dium through a Sep-Pak C 1 8 cartridge (Waters Chro-matography Div. , Milhipome Corp. . Milford, MA) by themethod described previously (1 5). The endothelin- 1

concentration in the medium was measured by RIAwith antibody against endothehin-1 and ‘251-labeled

endothelin- 1 as a tracer. In our preliminary study,heparmn did not interfere with our assay of endothe-

lin-1.

RNA Isolation

Total RNA from cultured EC was extracted by the

guanidmnium thiocyanate-phenol chloroform method(16). In brief, the cells were scraped, homogenizedwith 2 mL of solution buffer (4 M guanidlum thiocy-anate, 25 mM sodium citrate [pH 7.0J, 0.5% sarcosyl,

and 0. 1 M 2-mercaptoethanol). and subsequentlytransferred to a polypropylene tube. Sequentially, 0.2mL of 2 M sodium acetate, 2 mL of phenol (watersaturated), and 0.4 mL of chboroform-isoamyl alco-hal mixture (49: 1 ) were added to the homogenate,

with thorough mixing by inversion. The final suspen-sian was shaken vigorously for 1 0 s and cooled onice for 15 mm. Samples were centrifuged at 10.000

9 for 20 mm at 4#{176}C.After centnifugation, RNA in theaqueous phase was transferred to a fresh tube, mixedwith 2 mL of isopropanol, and then placed at -20#{176}C

for at least 2 h to precipitate RNA. Sedimentation at1 0,000 9 for 20 mm was again performed, and the

resulting RNA pellet was resuspended in 75%ethanol, sedimented, vacuum dried, and used as totalRNA.

Northern Blot Analysis for Endothelin-1 mRNA

Northern blot analysis for endothehin- 1 mRNA wasdetermined according to a method reported previ-

ously (17).Total RNA was denatured with 6% formaldehyde,

fractionated by 1 % agarose gel electrophomesis, and

transferred to a nylon membrane (Hybond-N; Amen-sham Japan, Tokyo. Japan). The membranes wereprehybridized at 42#{176}Cfor 5 h in a buffer containing50% fommamide, 5x Denhamdt’s reagent, 1 0% dex-tran sulfate, 5x sahine-sodium-phosphate-EDTA and1 00 �g/mL denatured single-strand DNA and werethen hybridized with 32P-labeled cDNA probes in thesame buffer at 42#{176}Cfor 1 6 h. After the filters werewashed, the hybridized RNA was visualized by ex-posume to Hyperfibm-TM (Amersham Japan) at

TABLE I . Effects of actinomycin D and heparin onendothelin-1 mRNA level in cultured aortic EC ofSHR#{176}

Endothelin- I mRNA Level(Absorbance)

a Growth-arrested aortic Et� of SHR were treated with or without

heparin (20 U/mL or 140 �g/mL) and actinomycin D (AD) (5 Mg/mL) atvarious periods, and the endothelln-1 mPNA level was quantitated bylaser densitometer and was standardized to �-actin mRNA. Data are

means of three experiments.

control

(pg/ 1 O� cells)

100

WKY

a)>a)

-J SHR

750

CG)

-C

� heparin 1OU/mL WKYC

w 1OU/mL SHR

24 (h)

Figure 1. Time-dependent accumulation of endothelin-1 in aortic EC culture medium of WKY and SHR with or without heparinat 10 U/mL (70 �g/mL). Data are means ± SD of five experiments. ‘ P < 0.001 compared with respective control level ofendothelin-1 (without heparmn). ‘ P < 0.05 and “ P < 0.01 compared with endothelin-I level of WKY with heparin at 10 U/mL(70 �g/mL).

0 6 12 18

Yokokawa et al

Journal of the American Society of Nephrology 1685

-70#{176}C. The membrane was prehybridized. hybrid-Ized, and washed as was the Northern blot. Theendothelin- 1 cDNA probe used in this study was

obtained from an EcoRI fragment of rat preproen-dothebin-1 cDNA (18). The probe was labeled with �a-

32PJdCTP by the multipnime DNA labeling system(Amersham Japan). After exposure to film, the probewas stripped by incubation in a buffer containing 5

mM TrIs-HC1 (pH 8.0), 2 mM EDTA, and 0. 1 x Den-hardt’s reagent at 70#{176}Cfor 1 h. The results werequantitated with a laser densitometer (Pharmacia

LKB Biotechnology AB, Uppsala. Sweden) and nor-

mabized to the constitutively expressed mouse fl-actin

mRNA signal.

Pharmacologic Treatment

The Endothelin-1 Level in Culture Medium. The

cultured EC of SHR and WKY were incubated withoutor with heparin at a dose of 10 U/mL (=70 �tg/mL).The endothehin- 1 content in the medium was meas-

ured at 6, 1 2, 1 8, and 24 h. EC were also incubatedwith various concentrations of hepanin ( 1 to 20 UImL or 7 to 140 �tg/mL) for 6 h, and endothehin-1levels in the medium were measured to determine thedose dependency of heparin’s effect.

Effect of Heparin on Endothelin-1 mRNA Expres-sion. The growth-arrested cultured aortic EC of WKYand SHR were exposed to serum-free DMEM with orwithout hepanin for 2 h. Total cellular RNA was

isolated, and the level of endothehin- 1 mRNA was

determined by Northern analysis.Actinomycin D Treatment. To know whether hep-

arm Inhibits endothehin- 1 mRNA expression at the

Control (60 mm) 0.20Control (30 mm) 0.10ADatTimeO 0.01AD at Time 30 mm 0.09Heparmn at Time 0 0.12Heparin at Time 30 mm 0.16Heparin + AD at Time 30 mm 0.09Heparin at Time 0 + AD at 0.06

TIme 30 mm

transcriptional level or posttmanscriptional level, we

have measured the endothehin- 1 mRNA bevel in cub-

tured EC of SHR after 1 h of incubation by using a

transcriptional inhibitor, actinomydin D (5 �zg/mL) (1)(see footnotes to Table 1).

Time Course of Endothelin-1 mRNA Expressionin the Presence of Heparin. The cultured aortic ECwere incubated without or with 1 0 U/mL (70 ,�g/mL)heparin for 1 , 2, 4, 8, 1 2, or 24 h. The endothelin- 1mRNA level was detected by automadiogmaphy andwas evaluated by laser densitometer.

(pg1’ 1 06 cells)

30

20

ic

a)>a,

-I

a,-C

0‘DC

w

0

Heparin. Inhibition of Endothelin Production . � . �, � � � � ,.� .� .� � , � �

1686 Volume 4 ‘ Number 9 ‘ 1994

Effect of Heparin on Agonist-Stimulated Endo-them-i mRNA Expression. The EC from SHR wereIncubated with angiotensin II (1 06 M) or TPA (10-6

M) for 2 h in the absence on presence of 10 U/mL (70�tg/mL) hepanin. The endothelin- 1 mRNA expressionwas detected by autoradiography. The membranes

were reprobed with mouse fl-actin cDNA.

Calculation and Statistical Analysis

The statistical significance of the results was eval-

uated by analysis of variance, and P values weredetermined by t test.

RESULTS

Blood Pressure

Mean blood pressures were measured by the tail-cuff method before the rats were euthanized, and thevalues were 107 ± 5.3 mm Hg for WKY weighing 256

± 10.0 g (N = 10) and 188 ± ii mm Hg for SHRweighing 250 ± 8.7 g (N = 10). We have obtained ECfrom 10 of each independent mat strain.

The Endothelin-1 Level in Culture Medium inthe Presence or Absence of Heparin

As shown in Figure 1 , immunomeactive endothehin-

1 was released from cultured EC from both WKY andSHR in a time-dependent manner. No significantdifference was observed in the basal level of endo-

thelln-1 in cultured medium between WKY and SHR.The addition of hepanin at a dose of 10 U/mL (70 �g/

mL) lowered the endothebin- 1 level in the culturemedium of both WKY and SHR. The suppression ofendothelin- 1 release with hepanin was significantlygreater in SHR than in WKY during the 6- to 24-hincubation periods. The endothehin- 1 content in themedium was decreased by the incubation with hep-

arm in a dose-dependent manner (1 to 20 U/mL or 7to 140 �g/mL) (Figure 2).

Effect of Heparin on Endothelin-1 mRNA Expres-sion

Heparin markedly inhibited the expression of en-

dothelin- 1 mRNA In EC from both WKY and SHR ina dose-dependent fashion (Figure 3). Densitometricmeasurements showed that the suppressive effect of

heparin on mRNA expression was more marked inSHR compared with WKY (Figure 4).

The results of the endothelin- 1 mRNA level in cub-

tured EC of SHR after 1 -hour incubation with acti-nomydin D (5 �g/mL) are presented in Table 1 . The

addition of actinomycin D at Time 0 resulted in al-most complete inhibition of the transcription of en-dothelin- 1 mRNA. When actinomydin D was added

at 30 mm. the endothehln-1 mRNA bevel measured

Figure 2. Dose-dependent effect of heparin on endothelin-I level in cultured medium of WKY and SHP. Data are means± SD of five experiments. Endothelin-1 level in culturedmedium was measured after 6 h of incubation. Heparin, IU/mi = 7 pg/mi. ‘ P < 0.001 compared with respectivecontrol level of endothelin-1. � ,� < 0.05 and � f� < 0.01compared with simultaneous values of WKY.

after another 30 mm (in an experiment of 1 h totalincubation) decreased compared with the controlbevel, indicating that actinomydin D arrests new RNAsynthesis after 30 mm. When hepanin (20 U/mL or

140 �g/mL) was added at 30 mm, the endothelin-1mRNA level measured after another 30 mm de-

creased compared with the control value. However,when hepamin was added with actinomydln D at 30mm, no further inhibition of endothebin- 1 mRNAlevel measured after another 30 mm (in 1 h totalincubation) was observed compared with thosetreated with just actinomydin D at 30 mm. Theseresults suggest that hepanin regulates the transcnip-tion mates of endothebin-1 mRNA.

Time Course of Endothelin-1 mRNA Expressionin the Presence of Heparin

The basal expression of endothebin- 1 mRNA wasroughly stable until the 8-h incubation period (Figure

Lull WKY

�

Heparin

Yokokawa et al

Journal of the American Society of Nephrology 1687

WKY

2.3kb-

18S- “�. 4

SHR

Endothelln-1

- Actk�

\;%%�OO �Q

� �.5,o ‘�

(i,, %, � ,�

b’?,- � *Q,, �

% % % %

\:���> %d’c�, �c:f.� ,.-

6_) 6� r��>

‘Figure 3. Effect of heparmn on endothelin-I mRNA expression. The growth-arrested cultured aortic EC of WKY and SHR wereexposed to serum-free DMEM with or without heparin for 2 h. Total cellular RNA was isolated, and the expression of endothelin-I mRNA was determined by Northern analysis. The membranes were reprobed with fl-actin cDNA.

Absorbance0.2 -

0.1 -

0(u/mi)

Figure 4. Evaluation by laser densitometer of the expressionof endothelin-1 mRNA (absorbance) with various concen-trations of heparin. Heparin, I U/mi = 7 �g/mL. Data aremeans of three experiments. Densitometric measurementsof three experiments showed that the suppressive effect ofheparin on mRNA expression (absorbance) was more ex-aggerated in SHR compared with that in WKY.

5). Thereafter, mRNA expression decreased in a time-dependent manner. On the other hand. hepamin at1 0 U/mL (70 �g/mL) suppressed endothehin- 1 mRNA

expression In a time-dependent manner, and the In-

hibitory effect of heparin was more augmented inSHR than In WKY.

Effect of Heparin on Agonist-Stimulated Endo-thelin-1 mRNA Expression

As shown in Figure 6, hepanin (10 U/mL am 70 �g/mL) suppressed both angiotensin II (1 06 M)- and TPA(1 0_6 M)-stimulated endothehin- 1 mRNA expression

in cultured aortic EC from SHR.

DISCUSSION

Endothehin- 1 , which has recently been isolatedfrom the supemnatant of cultured EC (6), exerts apotent vasoconstnictom effect on a variety of bloodvessels and produces a sustained elevation of sys-temic blood pressure (7). This study has demon-

strated that heparin suppresses the endothehin- 1peptide bevel and endothehin- 1 mRNA expression in

a dose- and time-dependent manner in cultured ECfrom SHR and WKY. These inhibitory effects of hep-arm in cultured EC were significantly more pro-nounced in SHR than In WKY. Thus, this study con-

E Control WKY

0 Control SHR

D Heparin-treated WKY(lOU/mi = 70�ig/mI)

#{149}Heparin-treated SHR(lOU/mI = 7Opg/ml)

(absorbance)

C 0.20

a

w

zccE 0.1

c

a

0�0Cw

0

TPA(106M)control heparin (lOU/mi

=7O,�g/ml)

A H �io6 M)control heparin

(10 U/mI=70 pg/mI)

Endothelin-118S-

Heparin. Inhibition of Endothelin Production

1688 Volume 4 ‘ Number 9 ‘ 1994

firms our previous observation that the release ofendothelin-i from cultured nat aortic EC is sup-pressed by hepanin (5) and further substantiates theobservation that hepanin affects mainly the expnes-sian of endothehln-1 mRNA in the process of endo-thehin- 1 synthesis. The endothehin- 1 mRNA expres-sian in cultured mat aortic EC was suppressed byhepanin. corresponding to a decrease in endothebin-1 peptide level in the medium. These findings to-gethem with those of actinomycin D experiments sug-gest that hepanin regulates endothehin-1 production,

probably at the bevel of transcription rates, althoughfurther nuclear run-on assays are necessary to rube

out a contribution from stability effects on mRNA.Several reports have shown the regulation of gene

expression by heparin at the transcriptional level.The effect of hepamin on gene expression is inde-pendent of ongoing protein synthesis (1). Heparininhibits c-fos mRNA ( 1 ) or tissue-type plasminogenactivator mRNA ( 1 9) at transcriptional levels. Hepa-rim reduced plasminogen activator inhibitor- 1 geneexpression in the presence of ECGS (20). The finding

of the greater inhibitory effect of hepanin on endo-thebln-1 mRNA expression in SHR than in WKY isinteresting but this observation requires further clam-ification.

Basal endothebin- 1 mRNA expression decreased astime passed (Figure 5). The basal endothelin- 1 me-leasing mate into the medium, as we can estimate by

the slope of graphs (Figure 1 ), also decreased come-spondingly. However, these changes in mRNAexpression do not always reflect the peptide bevel, iftheme are some step-up phenomena in the posttnan-

scriptional bevel in producing endothebin- 1. Accord-Ing to our previous study in endothelin-secneting tu-

mor, we could suggest step-up phenomenon in the

posttranscniptionab level in endothehin- 1 synthesis(17). Our previous studies have shown that basalendothelin- 1 release from cultured mat aortic EC intofreshly changed medium every 6 h is not decreased,even in the 24-h incubation period (5), in contrastwith these findings. One possibility to explain these

differences may be that several vasoactive sub-stances such as prostacyclin (2 1 ) and endothelium-

derived nitric oxide (22) or cytokmnes (23) produced

by EC may be involved in cellular function and/orcell growth, reflecting the production of endothehin-

1 . Abthough the endothehin- 1 mRNA expression doesnot require de rtovo protein synthesis (24), the post-

transcriptional mechanisms, including negativefeedback, in producing endothebin- 1 remain to beclarified.

Several experiments were performed to elucidate

the mechanism by which heparin inhibits geneexpression. Hepanin inhibits c-fos and c-myc mRNAexpression via a protein kinase C-mediated pathwayin calf (25) and rat ( 1 ) aortic VSMC and muninefibmoblasts (26). It also inhibits collagenase gene

expression mediated by a phorbol ester-responsiveelement in primate arterial smooth muscle cells (27).

These findings of the inhibition of both TPA- andangiotensin Il-induced endothehin- 1 mRNA expres-sion by hepanin (Figure 6) suggest the involvement ofprotein kinase C-mediated mechanisms in hepamin’sinhibitory effect on endothelin- 1 production. It has

been shown that both TPA and angiotensin II stim-ulate endothebin-1 production via protein kinase C-

dependent mechanisms (28,29). Taken together, hep-

arm may nonsebectively suppress protein kinase C-mediated gene expression, although further studiesare necessary to clarify this possibility.

In conclusion, hepanin suppresses endothebin- 1

4 8 12 24

Time (hours)

Figure 5. Time course of endothelin-I mRNA expression(absorbance) in the presence or absence of heparin at I 0U/mi (70 ,�g/mi) in cultured EC of WKY and SHR. The endo-thelin-I mRNA was evaluated by laser densitometer. ThemRNA optical density for each lane was standardized tothat of the constituently expressed �l-actin mRNA signal.Data are means of three experiments.

fl-actin #{149}#{149}I 0Figure 6. Effect of heparin on angiotensmn II (All)- or TPA-stimulated endothelin-1 mRNA expression in cultured ECfrom SHR. EC were incubated with agonists for 2 h in thepresence or absence of heparin at a dose of 10 U/mi (70�g/mL). Membranes were reprobed with �l-actin cDNA. Theposition of 18S ribosomal RNA is also shown. Angiotensin II,10_6 M; TPA, 10-6 M.

Yokokawa et al

Journal of the American Society of Nephrology 1689

production, probably at the transcriptional bevel.

These suppressive effects of heparin are more pro-

mounced In SHR than in WKY EC.

ACKNOWLEDGMENTS

This work was supported by the Uehara Memorial Foundation in

Japan and the U.S. Department of Veterans Affairs,

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