Supplementary Material � Online

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Supplementary Material – Online
Materials and Methods
Isolation of peripheral blood monocytes

Human peripheral blood mononuclear cells (PBMCs) were prepared from buffy coats (East

Anglian Blood Transfusion Service), screened for human immunodeficiency, hepatitis B and C

viruses. PBMCs were isolated by centrifugation over Ficoll-Hypaque and monocytes purified by

an adherence method as published18. PBMCs were added to gelatin-coated culture flasks and
incubated for 1 hours at 37oC. Non-adherent cells were removed by 3 washes of warm medium

(25-37oC). Adherent cells were then retrieved by brisk flushing with PBS containing 0.5mM

EDTA at 4oC. Cells were >90% viable as assessed with trypan blue exclusion. Cells were >99%

monocytes as assessed by flow cytometry with monocyte marker CD14. All media and reagents

used were endotoxin-free (<1ng/ml).

Adhesion itself may rapidly activate monocytes to differentiate into macrophages19. Thus

monocytes may have acquired macrophages properties after 1 day of adherent culture. However,

for clarity, the following terms will be used in this paper: monocytes describes CD14-postive

cells prior to adherence; macrophages in early culture (culture day1) will describe the same cells

after 1 day in culture; macrophages in late culture (culture day 8) will describe the same cells

after 8 days in culture.

Human vascular smooth muscle cells (VSMCs).

Human aortic VSMC (ASMCs) were obtained from donors from Addenbrooke's Hospital

Transplant Programme. Human plaque VSMCs were isolated from carotid plaques, removed at

carotid endarterectomy for cerebrovascular disease. Ethical Committee approval for use of

discarded tissue for research was obtained. Aortic or carotid plaque tissue was dissected under

sterile conditions and explant-cultured in 6-well dishes (Falcon) in M199 medium supplemented
with 20% FCS, antibiotics, L-glutamine and amphotericin. Histological examination of
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specimens confirmed that cells were medial (aortic) or intimal (carotid plaque) in origin. Cells

were subsequently passaged to approximately passage 4 (aortic VSMCs) or passage 1-2 (plaque

VSMCs).

In addition to primary cells, a non-immortalised human coronary artery VSMC cell line,

HCMED-1 E6, was used. These cells stably express the human papilloma virus E6 gene, which

results in extended life span, with reproducible cell proliferation and apoptosis over many

passages in culture20. We have recently extensively characterised the apoptotic responses of

these cells to cytokines and macrophages and demonstrated that they behave similarly to plaque-
derived VSMCs and aortic VSMCs7.

Detection of apoptosis in macrophage/VSMC co-cultures

VSMCs were seeded in 8-well chamber slides at 103 cells / well (plaque VSMCs) or 2x104 cells

/ well (HCMED-1 E6 cells) and incubated overnight. Monocytes were then added at varying

concentrations and co-cultures maintained for 8 days. Apoptosis was quantified by incubation of

co-cultures in 10 mg/ml propidium iodide (PI),(Sigma) and 125 g/ml bisbenzimide (Hoechst

33258; Sigma) for 10 min. Cells were then fixed in 400l 10% neutral-buffered formaldehyde

(Sigma) at 4°C, incubated with CD-14-FITC and analysed by fluorescence microscopy. The

amount of cell death was measured as the apoptotic index, calculated as:

Apoptotic Index = Dead Cells / (Dead Cells + Live Cells) x 100%

Regulation of apoptosis in co-cultures and VSMC cultures

To examine regulation of apoptosis in co-cultures, VSMCs were seeded as above but incubated

with 8x103monocytes / well for 8 days. NOS inhibitors were added at the start of macrophage

culture (L-Nitro-Arginine-Methyl-Ester (L-NAME) 100M, d-Nitro-Arginine-Methyl-Ester (d-

NAME) 100M, L-NG-MonoMethyl-Arginine (L-NMMA), d- NG-MonoMethyl-Arginine (d-

NMMA) (all from Sigma).

Griess assay for nitrite
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NO release from cultured human macrophages and VSMCs was measured using the Griess

colorimetric assay of nitrite, a stable inorganic end-product of NO produced by chemical reaction

with water. Human cells have much lower production of NO (and therefore nitrite) than rodent

cells. We therefore used a sensitive published modification of the Griess method of nitrite

determination for use with human macrophages21. This is accepted to have a lower limit of

detection of less than 1.0M21. Standard curves of sodium nitrite dissolved in culture medium
showed that A540 was linearly related to nitrite concentration between 1.0M and 500M, with

a lower limit of detectable nitrite concentration at 1.0M (not shown). Standard curves were
performed with every nitrite determination. To confirm that nitrite was derived from NO, nitrite

was measured in the presence and absence of NOS inhibitors.

Western blotting

Lysates of cultured VSMCs, monocytes or macrophages were prepared in non-reducing lysis

buffer (Tris pH 6.8, 4% SDS, 20% glycerol, 0.004% bromophenol blue), boiled for 5 minutes

and stored at -70oC. Protein concentration was estimated using the Lowry method (Biorad) and

10g/well electrophoresed using a 10% polyacrylamide gel. Proteins were transferred using

semi-dry blotting (Biorad) onto PVDF membranes (NEN). Membranes were incubated with

1/1000 anti-iNOS (mouse monoclonal, Transduction Laboratories) or 1/1000 anti-Fas (rabbit

polyclonal, Santa Cruz) in PBST (PBS, 4% non-fat milk protein (Marvel), 0.05% poly-oxy-

ethyl-sorbitan-mono-laureate (Tween 20)) at 4oC for 18 hours, washed 3x1 min and 4x5 min in

PBST, and incubated with sheep-anti-mouse-peroxidase or donkey-anti-rabbit-peroxidase-

conjugated antibodies (Amersham) in PBST for 2 hours at 25oC. Membranes were washed 3x30s

and 4x5 min and visualised with enhanced chemiluminescence (ECL, Amersham).
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Flow cytometry

For direct staining of mononuclear cells, cells were stained unfixed with FITC-conjugated

monoclonal antibodies at the manufacturer’s recommended concentration, or with isotype,

species and fluorescence ratio-matched control antibody (all antibodies from Sigma).

For flow cytometric analysis of expression of iNOS in VSMCs and monocyte/macrophages, 106

cells were suspended in 50l PBS containing primary antibody or isotype control (10g/ml anti-

TNF-, clone MAB210F, R&D Systems; 10g/ml anti-iNOS, Transduction Laboratories; or

control IgG1 MOPC31, Sigma) for 30 min at 4°C. Cells were then washed in PBS and incubated
with fluoresceinated sheep anti-mouse antibody (Sigma) for 30 min at 4oC. Finally, cells were

washed in PBS, post-fixed in 10% formaldehyde and analysed using a Becton Dickinson

FACScan flow cytometer within 24 hours. To analyse intracellular protein expression, cells were

fixed in 1% paraformaldehyde for 15 min at 4oC and permeabilised in 0.1% Triton-X-100 for 15

minutes at 4oC and then stained as before.

Immunocytochemistry

Thrombosed coronary arteries were collected from hospital autopsies for acute myocardial

infarction. There is Local Research Ethics Committee approval for immunohistochemistry on

these tissues. Tissues were paraffin-embedded and serially sectioned to identify plaque rupture

Sections of ruptured plaque were de-waxed and double-immunostained for iNOS and Fas-L.

Adjacent step sections were immunostained for CD68 (macrophage marker). Incubation with all

primary antibodies was for 24 h at 4oC. Sections were incubated in mouse monoclonal anti-iNOS

(1:100, Transduction Laboratories) and visualised with rabbit anti-mouse alkaline phosphatase

and Fast Blue BB (reaction product is Blue). Sections were incubated in polyclonal anti-Fas-L

(Santa Cruz) and visualised with biotinylated anti-rabbit (Dako), avidin/biotin/peroxidase (Dako)

and diaminobenzidine (Sigma) (reaction product is brown). To identify macrophages, sections
were incubated in mouse monoclonal anti-CD68 (1:50, clone KP-1, Dako) and visualised with

rabbit anti-mouse alkaline phosphatase and Fast Blue BB. Controls were mouse IgG1 and
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preimmune rabbit serum. Thus Fas-L was stained brown and iNOS and CD68 were stained blue.

To maximise interpretation of Fast Blue staining, sections were not counterstained and were

mounted in PBS/glycerol.
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Figure Legends For Online Figures
Figure I L-NAME and L-NIL inhibits macrophage-induced VSMC apoptosis in a dose-

dependent manner.

A. Human blood-derived macrophages were cocultured with human plaque VSMCs (passage 1)

for 8 days (Methods) and VSMC apoptosis measured with DNA-fluorochromes propidium

iodide and bisbenzimide (Methods). Cocultures were incubated in increasing concentrations of

L-NAME for 8 days. X-axis, L-NAME concentration (micromoles/litre (M), log scale. Y-axis,

VSMC Apoptotic Index (%).

B. Human blood-derived macrophages were cocultured with human HCMED1-E6 VSMCs

(Methods) and VSMC apoptosis measured with DNA-fluorochromes propidium iodide and

bisbenzimide (Methods). Cocultures were incubated in increasing concentrations of L-NIL for 8

days. X-axis, L-NIL concentration (micromoles/litre (M), log scale. Y-axis, VSMC Apoptotic

Index (%).

Figure II Aortic and HCMED1-E6 VSMC Fas is located intracellularly

A. Flow Cytometry. Non-immortalised (passage 6) human aortic VSMCs or HCMED1-E6

   VSMCs were cultured and fluorescein-stained for Fas for flow cytometry either

   permeabilised with detergent (total staining) or left unpermeabilised (surface staining)

   (Methods). X-axes, green fluorescence intensity (4 quadrant log scale); Y-axis, cell number.

   Leftward shaded histograms represent isotype-control staining (background); rightward open

   histograms represent specific fluorescence.

B. Double-immunofluorescence. HCMED1-E6 VSMCs were stained with rabbit polyclonal

   anti-60kDa-Golgi antibodies and anti-rabbit-FITC, and with mouse monoclonal anti-Fas and

   anti-mouse –Texas-red antibodies. For controls, isotype-control and preimmune rabbit serum

   were used and secondary antibody applied. Control fields are also shown in phase contrast
   illumination, to confirm cells are present.
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Figure III DETA/NO increases, and Brefeldin A inhibits, HCMED1-E6 VSMC Fas

expression

Flow cytometric analysis for surface Fas in VSMCs following incubation with DETA/NO alone

or with brefeldin A (5g/ml) or cycloheximide (10g/ml). All graphs: X-axis, green fluorescence

intensity (4 quadrant log scale); Y-axis, cell number. Leftward shaded histograms show isotype-

control staining (background); rightward open histograms show specific fluorescence.

Figure IV Effect of NO on Fas-induced apoptosis in VSMCs.

A. Apoptotic index of HCMED1-E6 VSMCs incubated either alone or in the presence of
   DETA/NO (1mM), agonistic anti-Fas antibody (400ng/ml) or in combination. Data are

   means, error bars represent S.E.M.s.

B. Apoptotic index of human plaque VSMCs incubated either alone or in the presence of

   DETA/NO (100M), agonistic anti-Fas antibody (400ng/ml) or in combination. Data are

   means, error bars represent S.E.M.s.

Figure V Effect of manipulating macrophage NO on surface Fas-L expression.

Top Row: macrophages (culture day 4) were incubated in the presence or absence of 100M

DETA/NO for 24 hours. Bottom row: macrophages (culture day 6) were incubated in the

presence or absence of 100 M L-NAME for 24 hours. All cells were non-permeabilised and

stained for surface Fas-L by immunofluorescence and analysed by flow cytometry (Methods).

All graphs: X-axis, green fluorescence intensity (4 quadrant log scale); Y-axis, cell number.

Leftward shaded histograms show isotype-control staining (background); rightward open

histograms show specific fluorescence.
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Figure VI Immunocytochemistry of ruptured coronary plaques for iNOS.

N=5 ruptured human coronary plaques were serially sectioned and adjacent sections

immunostained for iNOS, Fas-L and CD68 (Methods). Sections were double-immunostained for

iNOS (alkaline phosphatase, blue) and Fas-L (peroxidase, brown) (Methods). Adjacent sections

were immunostained for CD68 (alkaline phosphatase, blue)(Methods). One representative plaque

is shown. A low power view of an H&E section is shown for orientation (top) (x2 objective).

Higher power views (all x20 objective) are shown of the same macrophage-rich area in the

superficial cap.