Online Appendix for the following April 1 JACC article
TITLE: Hypoxia, Hypoxia-Inducible Transcription Factor, and Macrophages in Human
Atherosclerotic Plaques Are Correlated With Intraplaque Angiogenesis.
AUTHORS: Judith C. Sluimer, MSC, Jean-Marie Gasc, PHD, Job L. van Wanroij, MD, Natasja
Kisters, BsC, Mathijs Groeneweg, MSC, Maarten D. Sollewijn Gelpke, MSC, Jack P. Cleutjens,
PHD, Luc H. van den Akker, MD, Pierre Corvol, MD, PHD, Bradly G. Wouters, PHD, Mat J.
Daemen, MD, PHD, Ann-Pascale J. Bijnens, PHD
APPENDIX
Methods
THP-1 cell culture and differentiation. Human THP-1 cells were obtained from the American
Type Culture Collection (ATCC 10801, Manassas, Virginia). Cells were grown in RPMI
1640 medium (Gibco-Invitrogen, Grand Island, New York) containing 10% FetalClone III (v/v;
Hyclone, Pe-bio, Hogan, Utah), 1% GlutaMAX I (w/v; Gibco-Invitrogen), 125 mmol/l Hepes,
penicillin (100 units/ml), and streptomycin (100 µg/ml; Gibco-Invitrogen) at 37°C in a
humidified incubator with a 5% CO2 atmosphere. To differentiate THP-1 cells into macrophages,
cells were seeded at a density of 1  106 cells/ml and incubated for 72 h with 0.2 μmol/l phorbol
12-myristate 13-acetate (Sigma, Saint Louis, Missouri).
Microarray. Ribonucleic acid was isolated with the guanidine isothiocyanate/CsCl method
(Chomczynski, 1987 #326). Ribonucleic acid quantity and quality were determined with a
nanodrop spectophotometer (Witec AG, Littau, Switzerland) and a 2100 Bioanalyzer (Agilent
Technologies, Palo Alto, California), respectively. All samples included had a RNA Integrity
Number 5.
Double-stranded cDNA was synthesized from 1.6 or 2.0 μg total RNA using the OneCycle Target Labeling Kit (Affymetrix, Santa Clara, California), and used as a template for the
preparation of biotin-labeled cRNA with the GeneChip IVT Labeling Kit (Affymetrix). Biotinlabeled cRNA of 16 samples were hybridized to separate HGU133 2.0 Plus Arrays (Affymetrix),
washed, stained with phycoerythrin-streptavidin conjugate (Molecular Probes, Eugene, Oregon),
and the signals were amplified by staining the array with biotin-labeled anti-streptavidin antibody
(Vector Laboratories, Burlingame, California) followed by phycoerythrin-streptavidin. The arrays
were laser scanned with the GeneChip Scanner 3000 (Affymetrix) according to the
manufacturer's instructions. Data were saved as a raw image file and quantified using GCOS 1.2
(Affymetrix). Pre-processing and normalization of signal intensities was conducted with the error
model of Rosetta Resolver v5.1 (Rosetta Biosoftware, Seattle, Washington), which was
developed for Affymetrix GeneChips. Technical and biological duplicates were combined to
obtain intensities and standard errors per group. Rosetta Resolver and Locuslink (Entrez Gene)
were used to combine the expression intensities of multiple probes per gene to obtain the
expression intensity per gene. The error model of Rosetta Resolver was used to determine fold
changes between early and advanced stable atherosclerotic lesions and were considered
significantly different when p < 0.05. Ingenuity pathway analysis (Ingenuity Systems, Mountain
View, California) was performed on genes with a p < 0.01.
Online Table 1. Immunohistochemical Methods
Antibody
Company, Product Code
Control Tissue
Dilution
Incubation
Antigen
Blocking
Visualization
Retrieval
Pimonidazol
NPI, Hypoxyprobe
Normal skin
1 :50
1 h, RT
ChemMate
—
Powervision-HRP
HIF1a
Becton Dickinson, #610958
CCRA
1:120
O/N, 4°C
TE pH = 9
TBST/1%BSA
Powervision-HRP
Abcam, #463
CCRA
1:50
O/N, 4°C
—
TBST/1%BSA
Powervision-HRP
HIF2a
Abcam, #8365
CCRA
1:750
O/N, 4°C
ChemMate
TBST/1%BSA
Powervision-HRP
VEGF
Peprotech, #500-P10
ACA
1:50
O/N, 4°C
—
TBS/5%BSA
Powervision-HRP
GLUT1
Dako, #3536
COCA
1:100
1 h, RT
ChemMate
TBS/5%BSA
Powervision-HRP
GLUT3
Chemicon, #AB1345
Normal testis
1:500
1 h, RT
ChemMate
Biotin block
Powervision-HRP
HK1
Santa Cruz, #6517
Normal kidney
1:10
1 h, RT
—
TBST/1%BSA
Powervision-HRP
CD68
Dako, #M0814
ACA
1:100
30 min, RT
Pepsin
—
Powervision-HRP
CD31
Dako, #M0823
ACA
1:50
1 h, RT
TE pH = 8
TBS/5%BSA
Powervision-HRP/ABC-AP
CD34
Monosan, #MON1164
ACA
1:200
1 h, RT
TE pH = 8
TBS/5%BSA
Powervision-HRP/ABC-AP
Ki-67
Immunologic, #Rm9106-s
ACA
1:25
1 h, RT
TE pH = 8
TBS/5%BSA
ABC-HRP
Cleaved caspase 3
Cell Signaling, #9661
Tonsil
1:200
O/N, 4°C
ChemMate
TBS/5%BSA
ABC-HRP
ABC-AP/-HRP = alkaline phosphatase or horseradish-peroxidase conjugated avidin biotin complex (Vector laboratories); ACA =
atherosclerotic carotid artery; BSA = bovine serum albumin; CCRA = clear cell renal carcinoma; ChemMate = ChemMate target retrieval; COCA =
colon carcinoma; DAKO = Pepsin, 0.1% pepsin in 0.1N HCl; Powervision-HRP = horseradish-peroxidase conjugated Powervision goat anti-mouse
antibody (Immunologic); TBST = Tris-buffered saline with 0.1% Tween; TE = Tris-EDTA buffer. Other abbreviations are explained in the text.
Online Figure 1. No Pimonidazole Immunoreactivity in Control Patients
(A) No pimonidazole immunoreactivity was detected in the carotid atherosclerotic plaques of a
patient without pre-operative pimonidazole injection. Inset shows origin of magnification. (B) No
pimonidazole immunoreactivity was detected in the macrophages of carotid atherosclerotic
plaques of another patient without pre-operative pimonidazole treatment. Inset shows CD68
staining. (C) Hematoxylin and eosin staining of normal skin at incision site. (D) Hypoxia
(pimonidazole immunoreactivity) is present in the epithelium, hair follicle, and sebaceous gland
of normal skin tissue, a positive control for pimonidazole infusion.