Sahara et al.
ACE2 deletion accelerates vascular diseases 1
Supplementary Methods
Animals and Vascular Disease Models
ACE2 KO mice were generated from a germline chimera derived from mouse AB2.2-prime embryonic stem cells (Lexicon Genetics) with a targeted mutation (exon 3) of mouse ACE2 gene, as previously described
.
1 C57Bl6 and ApoE KO mice (C57Bl6 background) were purchased from Japan SLC and
Jackson Laboratory, respectively. Single knockout strains were backcrossed 10 times into a C57Bl6 background. ApoE/ACE2 double KO mice were generated by cross-breeding ApoE KO mice and
ACE2 KO mice, with genetic identity confirmed by tail genotyping at each generation. Male mice aged
8 to 10 weeks and weighing between 20 to 25 g in the littermates were used in this study: (1) wild-type
(WT); (2)
ACE2 KO; (3)
ApoE KO; and (4) ApoE/ACE2 double KO (n  10 per group).
To evaluate the impact of genetic ACE2 deletion on the development of vascular diseases, we used the two different experimental disease models: hyperlipidemia-induced atherosclerosis and mechanical injury-induced neointimal hyperplasia. In the former, ApoE KO and ApoE/ACE2 double KO mice were fed with cholesterol-rich western-type diet (0.15% cholesterol, 15% butter) and concurrently randomized to receive an AT1R blocker (ARB), olmesartan (Daiichi Sankyo Co., LTD.), at a dose of 1 mg/kg/d or vehicle by gavage every day for 12 weeks. In the latter, WT and ACE2 KO mice were anaesthetized by intraperitoneal injection of pentobarbital (50 mg/kg of body weight) and
underwent wire-mediated endovascular injury induced by inserting a straight spring wire (0.38 mm in diameter, No. C-SF-15-15,
COOK) into a femoral artery, as previously described.
2,3 Then, they were randomized to receive olmesartan (1 mg/kg/d) or vehicle by gavage every day for 4 weeks. Blood pressures and pulse rates were measured with a tail-cuff system (BP-98AW, Softron) every week.

Sahara et al.
ACE2 deletion accelerates vascular diseases 2
All experimental procedures and protocols were approved by the Institutional Committee for
Animal Research at the University of Tokyo and complied with the Guide for the Care and Use of
Laboratory Animals (National Institutes of Health (NIH) publication No. 85-23; revised 1996).
Quantification and Histological Analysis of Vascular Lesions
In the atherosclerosis study, 12 weeks after the initiation of feeding with cholesterol-rich diet, mice were euthanized by intraperitoneal injection of an overdose of pentobarbital and perfused with 0.9% sodium chloride solution. Both the heart and whole aorta were immediately harvested. After adventitial fatty and collagen tissues were removed, the aorta was excised and opened longitudinally and fixed with 4% paraformaldehyde. To quantify the atherosclerotic lesions, the thoracic aorta was then analyzed with en face Sudan IV staining.
4
Sudan IV-positive (red) lesion area and total aortic area were measured using imaging analysis software (Image J, NIH). Separately, aortas were also collected and snap frozen and stored at

80

C for subsequent RNA and protein extraction. Next, to assess the atherosclerotic lesions in aortic root, the heart was cut in the plane between the lower tips of the right and left atria. The upper portion was snap frozen in OCT compound (TissueTek). The aortic root was sectioned serially (5
 m intervals) from the appearance of the aortic valve to the ascending aorta until the valve cusps were no longer visible. Frozen sections were then analyzed with elastic Van Gieson (EVG) and Oil red O staining to detect the atherosclerotic lesions in aortic root and lipid deposition in the lesions.
In the neointimal hyperplasia study, 4 weeks after surgery, mice were euthanized by intraperitoneal injection of
an overdose of pentobarbital and perfused with 0.9% sodium chloride solution. The injured femoral arteries were immediately harvested, fixed in 4% paraformaldehyde and embedded in paraffin.
Paraffin-embedded sections (5
 m) were then analyzed with hematoxylin and eosin (HE) and EVG

Sahara et al.
ACE2 deletion accelerates vascular diseases 3 staining for the measurement of lesion areas. The parts of injured arteries were snap frozen and stored at

80

C for subsequent RNA and protein extraction.
Immunohistochemistry
Frozen sections of aortic root and paraffin-embedded sections of femoral artery were processed for immunohistochemical staining for examination under light microscopy. Immunohistochemical analyses involved the incubation of the sections with the anti-mouse primary antibodies, including anti-

-smooth muscle actin (

SMA; Sigma-Aldrich), anti-monocyte/macrophage marker (MOMA)2 (Serotec), anti-MMP9 (Santa Cruz) and anti-ACE2 (R&D) antibodies, followed by incubation with a biotinylated secondary antibody (Dako) using the avidin-biotin complex technique with Vector Red substrate
(Vector Laboratories). The nuclei were counterstained with hematoxylin.
Quantitative Real-time RT-PCR
Total RNA was isolated from the aorta and femoral artery using RNeasy mini extraction kit (Qiagen) and reverse-transcribed with a moloney murine leukemia virus reverse transcriptase using a cDNA synthesis kit (ReverTra Ace-

; Toyobo). Quantitative real-time RT-PCR was performed on Mx 3000P
QPCR System (Stratagene) using SYBR Green real-time PCR Master Mix (Toyobo) for 40 cycles.
Standard deviations of the means in quantitative RT-PCR experiments were obtained from three independent experiments. The primer sequences for the detection of vascular inflammation-related genes of mice and humans, including VCAM-1, intercellular adhesion molecule (ICAM)-1, MCP-1,
TNF

, interleukin-6 (IL-6), IL-10, RANTES, IL-1

, macrophage inflammatory protein (MIP)-1

,
MIP-2

, MMP2 and MMP9, are listed in Supplementary Table S1 . Thresfold cycles of primer probes

Sahara et al.
ACE2 deletion accelerates vascular diseases 4 were normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and translated to relative values.
Angiotensin II Peptide Studies
At sacrifice, plasma was collected from each mouse. The isolated aorta or femoral artery was homogenized in a lysis buffer containing a protease inhibitor cocktail (Sigma-Aldrich). Ang-II concentrations in plasma and aorta/femoral artery extracts were measured by a commercial radioimmunoassay using two antibodies specific for Ang-II (SRL, Inc.).
5
Measurement of MCP1 and Soluble VCAM-1 Levels
Concentrations of MCP1 and soluble VCAM-1 proteins in aortic tissue extracts or the cell media as indicated below were measured by enzyme-linked immunosorbent assay (ELISA; R&D Systems), according to the manufacturer’s instructions.
Western Blotting
Proteins from the aorta/femoral artery tissues or cultured cells as indicated below were extracted after homogenization in a lysis buffer containing a protease inhibitor cocktail, as previously described.
6
The protein samples (5
 g) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) and transferred onto a polyvinylidene fluoride membrane (Hybond-P; GE Healthcare).
The membranes were incubated with anti-mouse primary antibodies to ACE2 (Sigma), extracellular signal-regulated kinase ( ERK)1/2, phospho-ERK1/2, JNK1/2, phospho-JNK1/2, p38 mitogen-activated protein kinase (p38 MAPK) and phospho-p38 MAPK
(Cell Signaling Technology), followed by

Sahara et al.
ACE2 deletion accelerates vascular diseases 5 incubation with a horseradish peroxidase-conjugated secondary antibody. Next, an enhanced chemiluminescence system (ECL Plus; GE Healthcare) was used to detect immunoblotting, and bands were visualized and quantified with a lumino-analyzer (LAS-1000; Fujifilm). The signal intensity was normalized to

-actin expression.
Cell Studies with Isolated Murine and Human Macrophages
Primary macrophages were isolated from the bone marrow of WT (C57Bl6) and ACE2 KO mice.
7
Briefly, mice were euthanized by intraperitoneal injection of an overdose of pentobarbital, and the femur and tibia were harvested and placed on ice. The marrow from the bone was flushed with a 1 ml syringe with a 27-gauge needle. After hemolytic reaction and centrifugation at 1000 rpm for 5 min, cell pellets were resuspended in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin and cultured in T75 ml flasks, in which fibroblasts adhered on the bottom. The solution including non-adherent macrophages was decanted into 10 cm petri dishes on the following day, and macrophages were cultured and adhered over a week. The lineage of macrophages was confirmed by the expression of CD14 and staining for MOMA-2. The adherent macrophages were scrapped off, transferred into 6-well plates, and incubated in RPMI 1640 medium for 48 h. Then, cells were stimulated with TNF

(1 ng/ml; R&D) for 4 h in serum-free RPMI 1640 medium . Total RNA of cultured cells and the culture media were harvested. Pro-inflammatory responsiveness was evaluated by the changes in mRNA expression of VCAM-1, MCP-1, IL-6, RANTES and MMP9 genes, measured with quantitative RT-PCR, and also by the secretion of MCP-1 protein into the media, measured with
ELISA. Recombinant human ACE2 (rhACE2) protein (R&D) was used for the rescue experiments with the macrophages derived from
ACE2 KO mice.

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ACE2 deletion accelerates vascular diseases 6
A human monocytic cell line, THP-1 cells were purchased from the American Type Culture
Collection (Manassas, the United States). THP-1 cells were cultured on 6-well plates in
RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, as previously described.
8 THP-1 cells were differentiated into macrophage-like cells by treatment with 200 nM
12-O-tetradecanoyl-phorbor-13-acetate (TPA; Sigma) for 48 h.
9 Differentiated THP-1 cells were investigated in the small interfering RNA (siRNA) transfection study as below.
Cell Studies with Murine and Human Aortic Smooth Muscle Cells
Primary vascular smooth muscle cells (VSMCs) were isolated from the aorta of WT (C57Bl6) and
ACE2 KO mice that were euthanized by intraperitoneal injection of an overdose of pentobarbital, as previously described, 10
and cultured in smooth muscle growth medium (SmGM)-2 (Lonza; Basel,
Switzerland). Cells were then stimulated with Ang-II (10
-8
or 10
-7
M; Sigma-Aldrich) for 60 min in serum-free DMEM. Total RNA of cultured cells and the culture media were harvested.
Pro-inflammatory responsiveness was evaluated by changes in mRNA expression of VCAM-1, MCP-1,
IL-6, RANTES and MMP9 genes, measured with quantitative RT-PCR, and also by the secretion of soluble VCAM-1 protein into the media, measured with ELISA. Phosphorylation status of ERK1/2,
JNK1/2 and p38 MAPK in the VSMCs treated with Ang-II was assessed by Western blotting. To determine which signaling pathway mediates angiotensin II-induced pro-inflammatory responsiveness, primary VSMCs were pretreated with PD98059 (ERK inhibitor; 10

M) (Calbiochem), SP600125
(JNK inhibitor; 10

M) (Sigma-Aldrich), or SB203580 (p38 MAPK inhibitor; 10

M) (Calbiochem) for 60 min prior to the Ang-II stimulation. After the following treatment with Ang-II for 60 min, pro-inflammatory responsiveness was determined as above.

Sahara et al.
ACE2 deletion accelerates vascular diseases 7
Human aortic smooth muscle cells (HASMCs) were purchased from Lonza. They were grown in
SmGM-2 and used in passages 5

9 for the siRNA transfection study as below.
Transfection of Small Interfering RNA against ACE2 and JNK1/2 into Human Macrophages and HASMCs
A siRNA duplex against human ACE2 was designed by Ambion and its sequence was
5’-GGCCAUUAUAUGAAGAGUATT-3’. The target sequence for JNK1/2-specific siRNA is
5’-AAAAAGAATGTCCTACCTTCT-3’, which is specific for JNK1/2 of both mice and humans, 11 and the siRNA duplex against JNK1/2 was synthesized by Invitrogen. Differentiated THP-1 cells into macrophage-like cells (2  10 5 cells per well in 6-well plates) were incubated with ACE2-specific or scrambled control siRNA (30 nM) using a transfection agent (Lipofectamine 2000; Invitrogen) for 4 h, and then fresh
RPMI 1640-based medium was added. Gene silencing of ACE2 was monitored after 48 h by quantitative RT-PCR using the extracted total RNA. Then, cells were stimulated with
TNF

(1 ng/ml) in the absence or presence of rhACE2 for 4 h. Total RNA of cultured cells were harvested for expression analyses of the inflammation-related genes ( Supplementary Table S1 ).
HASMCs were transfected with ACE2-specific or scrambled control siRNA (30 nM) using
Lipofectamine 2000. After 48 h of incubation in SmGM-2 supplemented with 2% fetal bovine serum, cells were stimulated with Ang-II (10
-7
M) for 60 min in serum-free SmGM-2. Using the extracted total
RNA, pro-inflammatory responsiveness was determined by changes in mRNA expression of the inflammation-related genes as above. Meanwhile, isolated primary VSMCs were transfected with
JNK1/2-specific or scrambled control siRNA (30 nM) using Lipofectamine 2000. After 48 h of incubation, cells were stimulated with Ang-II (10
-7
M) for 60 min and pro-inflammatory responsiveness

Sahara et al.
ACE2 deletion accelerates vascular diseases 8 was determined as well.
NF-

B Transcriptional Factor Assay
The NF B activity in human
THP-1 cells before and after treatment with
TNF
 in the absence or presence of genetic ACE2 knockdown
was measured using a commercially available kit (
NF B (p50)
Transcription Factor Assay Kit; Rockland Immunochemicals, Inc.
). First, differentiated THP-1 cells into macrophage-like cells were transfected with ACE2-specific or scrambled control siRNA. After 48 h, cells were stimulated with
TNF

(20 ng/ml) for 60 min, and then cellular nuclear extracts were isolated.
Nuclear extracts were put on a 96-well plate, where specific oligonucleotides containing the
NF B response element (5’-GGRNNYYCC-3’) were immobilized onto the bottom of wells. The active complex form of NF B contained in nuclear extracts specifically binds to the NF B response element.
This combined NF B (p50) was detected by a specific primary antibody directed against NF B (p50).
A secondary antibody conjugated to HRP was used to provide a sensitive colorimetric readout at 450 nm by spectrophotometry.
Absorbance data were translated to relative values.
Statistical analysis
Data are presented as the mean ± standard deviation (SD). The comparison of the means was performed by a one-way analysis of variance (ANOVA) followed by Scheffé’s post hoc test. Statistical significance was defined as P <0.05.

Sahara et al.
ACE2 deletion accelerates vascular diseases 9
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ACE2 deletion accelerates vascular diseases 10
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