Supplemental Methods

Immunoblotting

Protein lysates (30 µg protein) were and separated by SDS gel

electrophoresis and transferred to nitrocellulose membranes as described previously 1.

Membranes were exposed to a rabbit antibody against rabbit-15-LO-1 (Caymen

chemicals) (dilution 1:5000 in TBS) for 1 h at room temperature, rinsed with TBS buffer

containing 0.1% Tween-20 and incubated with a 1:5000 diluted goat anti-rabbit IgG

(horseradish peroxidase conjugated, Zymed). Membranes were re-probed with mouse

anti-β-actin as a loading control. Immunoreactive bands were identified and

densitometric analysis was performed.

Quantitative real time polymerase chain reaction (qRTPCR)

Total nucleotides were isolated from rabbit arteries using Trizol reagent (Life

Technologies). The DNA was digested with amplification grade DNase-1 (Invitrogen).

cDNA was synthesized from total RNA by using SuperScript III first strand synthesis

system for qRTPCR (Invitrogen). For control, one reaction mixture without reverse

transcriptase and one without RNA was made. cDNAs for 15-LO-1 and GAPDH in the

reaction mixtures was amplified using BIO-Rad iCycler in a 25 µL mixture containing; 2

X 10-7 g cDNA, 2 X 10-7 M primers and RT² SYBR Green / Fluorescein qPCR Master

Mix (Super Array Bioscience Technologies). The primers for 15-LO-1, (reverse) 5’- CCT

GGC GCG GAC GTT GAT CTC-3’ and (forward) 5'- TGG CTG CCC CGC TGG TCA

TGC-3', were designed from the cDNA sequence of rabbit 15-LO-1, and were

synthesized by the Operon Co (Huntsville, AL). The primers for GAPDH were supplied

by Super Array Bioscience Technologies. The program for the iCycler was 94°C for 30 s,

58°C for 1 min, and 72°C for 1.5 min, repeated 40 times followed by final extension at

72°C for 7 min. The PCR products obtained after the iCycler amplification were

separated by 1% agarose gel electrophoresis and visualized by ethidium bromide staining

to confirm the presence of a single amplified product.

Immunohistochemistry

Rabbit aortic or mesenteric arterial segments were fixed in 4% paraformaldehyde

and cut into sections as described previously 1, 2. Sections were incubated with mouse

anti-human platelet endothelial cell adhesion molecule (PECAM) (kindly provided by Dr.

Peter Newman, Blood Center for Southeastern Wisconsin, Milwaukee, WI) and rabbit

anti-human 15-LO-1 diluted 1:500 in 0.2% Triton X-100 containing 1% normal goat

serum. Sections were incubated for 1 h at room temperature, rinsed, and incubated

with 1:500 anti-mouse Alexa-Fluor 594 and anti-rabbit FITC labeled secondary

antibodies (Molecular Probes) for 1 h at 25°C. Sections were mounted with media

containing 1% 4,6-diamidino-2-phenylindole (DAPI) and protected by a glass coverslip.

Fluorescent images were captured (200X magnification) using Nikon Eclipse E600

microscope and Spot Advanced software.

Metabolism of 14C-AA

Arteries were dissected, cleaned and cut into 2-3 mm rings in N-2-

hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (mM): 10 HEPES,

150 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 6 glucose, pH 7.4. Rings were incubated at 37°C

with indomethacin (INDO) (10-5 M) (Sigma, MO) or with INDO and BW755C (5 X

10-5 M) (Burroughs Wellcome, Sandwich, England) in 5 ml HEPES for 10 min and

then [14C]-AA (0.5 µCi, 10-7 M) was added. After 5 min, A23187 (10-5 M)

(Sigma, MO) was added. After 15 min, the reaction was stopped with ethanol (15% final

concentration), and the samples were extracted using Bond Elute octadecylsilyl columns3.

The extracts were analyzed by reverse phase high-pressure liquid chromatography

(HPLC) using a Nucleosil C-18 (5µ, 4.6 x 250 mm) column. The solvent system

consisted of a 40 min linear gradient (flow rate=1 ml/min) from 50% solvent B

(acetonitrile with 0.1% glacial acetic acid) in solvent A (deionized water) to 100%

solvent B. Column effluent was collected in 0.2 ml fractions, and the radioactivity was

determined.

Isometric tension recording

Aortic rings were suspended in a 6 ml tissue bath with Krebs buffer of

composition: (mM) 119 NaCl, 4.7 KCl, 1.8 KH2PO4, 1.17 MgSO4, 25 NaHCO3, 2.5

CaCl2, 0.026 EDTA, and 5.5 glucose bubbled with 95% O2 and 5% CO2 at 37°C.

Isometric tension was measured with force-displacement transducers using glass muscle

baths and recorded with a Macintosh computer and MacLab software. The aortic rings

were gradually adjusted to the resting tension of 2gm and allowed to equilibrate for 1 h.

The rings were then tested for the maximum response with 44 mM KCl. The rings were

treated with INDO (10-5 M) and LNA (3x10-5 or 3x10-4 M) for 20 min. In some studies,

the aortic rings were pretreated with CDC (20x10-5 M), BW755C (5x10-5 M), ebselen

(20x10-5 M), APA (10-7 M), CTX (10-7 M), or TRAM-34 (10-5 M) either alone or in

combination with each other. These inhibitors were dissolved in a proper solvent and

added to the muscle bath along with INDO and LNA. After 20 mins of incubation with

the inhibitors, arteries were contracted by PHE (approx. 10-7 M) to 50-60% of the

maximal KCl contraction and cumulative concentrations of AA (10-7-10-4 M) or ACH

(10-9-10-5 M) were added to the bath and changes in isomeric tension were measured.

Relaxations to EBIO, P1075 and DPTA: Arteries were prepared in the Krebs

buffer as described above. Contractions to KCl (44mM) or PHE were determined,

precontracted with PHE to half maximal contractions and relaxations to cumulative doses

of IKCa-channel opener 1-ethyl-2-benzimidazolinone (1-EBIO; 10-8-10-3 M), KATP-

channel opener P1075 (10-8-10-3 M) or NO-donor dipropylenetriamine NONOate

(DPTA;10-9-10-5 M) were measured. In some experiments the arteries were treated with

INDO, LNA, CTX, ebselen or BW755C either alone or in combination with each other

for 20 mins and then the relaxations were determined.

Measurement of membrane potential

Membrane potential (Em) was measured in the SMCs of the rabbit aortas as

published previously 5. Briefly, aortic rings were cut open laterally, pinned to a silastic

layer in a perfusion chamber, with the endothelium exposed and perfused with warmed

(37 0C) Krebs buffer bubbled with 95% O2 and 5% CO2. After 30 min of equilibration, Em

of SMCs was measured either in presence of vehicle, INDO (10-5 M), INDO and PNE (l0-

7 M), INDO and PNE plus BW755C (10-4 M) or BW755C alone. Impalements were done

with a glass electrode only in a small section of the aortas where the endothelium was

removed by gentle rubbing with a small cotton swab. Glass microelectrodes were filled

with 3 M KCl and had estimated tip size of 0.1 – 0.2 µm, tip resistance of 30 – 80 MΩ,

and tip potentials of ≤3 mV. Electrodes were attached to high-impedence biological

amplifier (Dagan Cell Explorer, Dagan Instruments; Minneapolis, MN). Electrode

polarization was eliminated by a Ag/AgCl half cell. Criteria for a successful impalement

included (a) an abrupt drop in potential to a new steady-state value, which was

maintained for a minimum of 5 s, (b) Em value greater than -20 mV, and (c) an abrupt

return to the original baseline when the electrode was retracted from the tissue. AA (10-5

M) was added to the aortic segments, and after 10 min Em was measured.

Statistical analysis

The experimental data were expressed as means ± S.E.M. A repeated measure

two-way ANOVA followed by Bonferronni post-test was performed to analyze responses

to each ACH or 1-EBIO cncentration and effect of inhibitors on these responses. Values

were considered significant at p<0.05 or smaller.

References to Online Supplement

1. Aggarwal NT, Holmes BB, Cui L, Viita H, Yla-Herttuala S, Campbell WB. Adenoviral expression of 15-lipoxygenase-1 in rabbit aortic endothelium: role in arachidonic acid-induced relaxation. Am J Physiol Heart Circ Physiol 2007;292(2):H1033-41.

2. Tang X, Spitzbarth N, Kuhn H, Chaitidis P, Campbell WB. Interleukin-13 upregulates vasodilatory 15-lipoxygenase eicosanoids in rabbit aorta. Arterioscler Thromb Vasc Biol 2003;23(10):1768-74.

3. Pfister SL, Spitzbarth N, Nithipatikom K, Edgemond WS, Falck JR, Campbell WB. Identification of 11,14,15- and 11,12,15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta. J. Biol. Chem. 1998;273:30879-30887.

4. Campbell WB, Spitzbarth N, Gauthier KM, Pfister SL. 11,12,15-Trihydroxyeicosatrienoic acid mediates acetylcholine-induced relaxations in the rabbit aorta. Am J Physiol Heart Circ Physiol. 2003;285(6):H2648-2456.

5. Chawengsub Y, Aggarwal NT, Nithipatikom K, Gauthier KM, Anjaiah S, Hammock BD, et al. Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta. Am J Physiol Heart Circ Physiol 2008;294(3):H1348-56.

Legends to Supplement Figures Figure S1. Immunohistochemical localization of 15-LO-1 in arteries from

hypercholesterolemic or normal rabbits. Arterial sections were co-stained for platelet

endothelial cell adhesion molecule (PECAM), 15-LO-1, and nucleus. For controls

sections were either not stained (panel E & M) or stained with only secondary antibodies

(panels A & I). 15-LO-1 expression is localized to the endothelium (white arrow).

Figure S2. Agonist-induced relaxations in aortas from normal or hypercholesterolemic

rabbits. (A) Aortas were precontracted with phenylephrine (PNE) and relaxations to

cumulative doses of acetylcholine (ACH) were determined. (B) Aortas from normal

rabbits were incubated with indomethacin (INDO; 10-5 M) and L-nitroarginine (LNA; 3

X 10-5 M or 3 X 10-4 M), precontracted with PNE and relaxations to ACH were

determined. (C and D) Aortic rings were incubated with INDO (10-5 M), precontracted

with PNE and relaxations to ( C) a nitric oxide-donor, dipropylenetriamine NONOate

(DPTA) , and (D) a KATP-channel opener, P1075, were determined. Values are expressed

as percentage mean �± SEM. *** = p<0.001, ** = p < 0.01, * = p<0.05.

Figure S3: Relaxations in aortic rings from normal rabbits. Aortic rings were pretreated

with indomethacin (INDO, 10-5 M), L-nitroarginine (LNA, 3 X 10-5 M) and either

vehicle, BW755C (5 X 10-5 M), ebselen (2 X 10-5 M), or CDC (2 X 10-5 M). Rings were

than precontracted with phenylephrine (PNE, 10-7 to 10-6 M) and relaxations to (A)

acetylcholine (ACH) or (B) arachidonic acid (AA) determined. Values represent mean

±SEM. (*** = p<0.001, * or # = p<0.05). * BW755C treatment, # ebselen treatment.

Figure S4: Relaxations in aortic rings. Aortic rings were pretreated with indomethacin

(INDO, 10-5 M), L-nitroarginine (LNA, 3 x 10-5 M) and vehicle or various inhibitors,

precontracted with phenylephrine (PNE, 10-7 to 10-6 M) and relaxations to acetylcholine

(ACH) determined. (A) Relaxations in aortic rings from hypercholesterolemic rabbits

incubated with INDO, LNA and BW755C (5 X 10-5 M) either alone or in combination

with charybdotoxin (CTX, 10-7 M) or TRAM-34 (10-5 M). Relaxations in aortic rings

from hypercholesterolemic rabbits (B) or normal rabbits (C) incubated with INDO, LNA

and CTX or TRAM-34. Values represent mean ±SEM. (*** or ### = p<0.001, ** or ## =

p<0.01, * or # = p<0.05).

Figure S5: Relaxations to 1-ethyl-2-benzimidazolinone (1-EBIO) in aortic rings from

normal or hypercholesterolemic rabbits. Aortic rings were pretreated with indomethacin

(INDO, 10-5 M), L-nitroarginine (LNA, 3 x 10-5 M), precontracted with phenylephrine

(PNE, 10-7 to 10-6 M) and relaxations to 1-EBIO determined (A). Aortic rings from

hypercholesterolemic rabbits were treated with INDO, LNA and either vehicle,

charybdotoxin (CTX,10-7 M), BW755C (5 X 10-5 M) or ebselen (2 X 10-5 M),

precontracted with PNE and relaxations to 1-EBIO determined (B). Values represent

mean ±SEM. (** = p<0.01, * = p<0.05).

Figure S 1

Figure S 2

10-10 10-9 10-8 10-7 10-6 10-5

0

20

40

60

80

100

120Cholesterol-fedNormal

**

*

ACH (M)

10-10 10-9 10-8 10-7 10-6 10-5

0

20

40

60

80

100

120

INDOINDO+30µM LNAINDO+300µM LNA

******

ACH (M)

10 -9 10 -8 10 -7 10 -6 10 -5

0

20

40

60

80

100

120

NormalCholesterol-fed

DPTA (M)10-9 10-8 10-7 10-6 10 -5 10-4

0

20

40

60

80NormalCholesterol-fed

P1075 (M)

A B

D C

10-8 10 -7 10-6 10 -50

25

50

75

100 INDO + LNA

INDO + LNA + CDCINDO + LNA + EbselenINDO + LNA + BW

ACH (M)

10-7 10-6 10 -5 10 -4 10-30

25

50

75

100

******

INDO + BWINDO

AA (M)

A

B

Figure S 3

* #

Figure S 4

10-9 10-8 10-7 10-6 10-5

0

25

50

75

100

INDO + LNA + TRAM + BW

INDO + LNA

INDO + LNA + CTX + BW

INDO + LNA+ BW

ACH (M)

10-9 10-8 10-7 10-6 10-5

0

25

50

75

100 INDO +LNAINDO + LNA + CTXINDO + LNA + TRAM

ACH (M)

******

###

###

*

#

10-9 10-8 10-7 10-6 10-50

25

50

75

100 INDO + LNAINDO + LNA + CTXINDO + LNA + TRAM

**#

#

ACH (M)

### *** ***

A

B

C

A

B

Figure S 3

10-8 10-7 10-6 10-5 10-4 10-3

0

25

50

75

100 Cholesterol-fedNormal

1-EBIO

10-8 10-7 10 -6 10-5 10-4 10 -3

0

25

50

75

100 INDO + LNA

INDO + LNA + CTXINDO + LNA + BW

INDO + LNA + Ebselen

**

***

1-EBIO (M)

Figure S 5