Online Data Supplement
Methods
Reagents
Antibody against phospho-VEGF receptor 2 (p-VEGFR2; Tyr996) was purchased from Cell
Signaling Technology (Beverly, MA, USA). Acetylcholine chloride, atropine sulfate,
mecamylamine hydrochloride, methyllycaconitine citrate, pyridostigmine bromide, and
antibodies against vesicular acetylcholine transporter (VAChT), nAChR 7 subunit
(7-nAChR), peroxidase-conjugated -actin, and peroxidase-conjugated second antibody
against rabbit/rat were obtained from Sigma-Aldrich (St. Louis, MO, USA). Ketanserin was
obtained from Janssen Pharmaceutica (Beerse, Belgium).
Immunohistochemical Studies of Capillary Density
Animal subjects were perfused with a 0.9% NaCl solution followed by a 4%
paraformaldehyde in 0.1 mol/L phosphatebuffer (pH 7.4). The heart was fixed in 4%
paraformaldehyde for 24 h. Sections (5 μm) were cut transversely at 200 μm intervals into 5
slices from the ligation site to the apex. Sections were washed, dehydrated in a graded
ethanol series and embedded in paraffin. Capillary density examined using an antibody
against CD34 for rat (R&D Systems, San Diego, CA, USA; 5 μg/ml) or mouse (Abcam,
Cambridge, UK; 2 μg/ml). Myocytes were counterstained with eosin. Capillary density for
each section was determined in ten randomly selected fields and is expressed as numbers of
capillary/field (400×).
Isolation, Identification and Culture of Cardiac Microvascular Endothelial Cells
(CMECs)
Rat heart was removed and perfused with 10 ml calcium-free PBS. The outer one-fourth of
the left ventricle wall and septum was dissected away to remove epicardial arteries and larger
penetrating vessels. The remaining tissue was minced in 0.2% type II collagenase (Invitrogen,
Carlsbad, CA, USA) and incubated for 40 min at 37°C in a shaking bath. Trypsin (0.02%;
Invitrogen, Carlsbad, CA, USA) was then added, and the tissue was sheared 10 times over a
period of 30 min. Dissociated cells were filtered through a 100-µm mesh filter, washed with
DMEM, and centrifuged at 1,200 g for 5 min. CMECs were resuspended in DMEM
supplemented with 20% FBS and antibiotics (penicillin 100 IU/ml and streptomycin 100
mg/ml). The CMECs were seeded in 1% laminin-coated flask. After a 2-h period, attached
cells were washed with DMEM to allow differential adhesion and cultured in DMEM with
20% FBS in a humidified atmosphere of 5% CO2 at 37°C. CMECs were grown to confluency
(6-7 days) and used after first passage. After isolation and plating on culture plates for 6 days,
cells were incubated with 10 μg/ml DiI AcLDL (Molecular Probes, Eugene, OR, USA) in
DMEM containing 20% FBS overnight. After washing three times with PBS, cells were fixed
with 2% paraformaldehyde for 30 minutes, incubated with a goat antibody against CD34
(R&D Systems, San Diego, CA, USA; 5 μg/ml) for 1 hour, and incubated with FITC-labeled
anti-goat IgG (Sigma-Aldrich, St. Louis, MO, USA; 1:200) for 30 minutes. Cells positive for
both DiI AcLDL and CD34 were judged to be CMECs. The culture derived as such
contained >95% CMECs (Figure 3S).
Western Blotting
CMECs were lysed with a RIPA buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1%
Nonidet P-40; 0.1% SDS; and 0.5% sodium deoxycholate) and tissue membrane proteins
were extracted with Mem-PER Eukaryotic Membrane Protein Extraction Reagent Kit
(Thermo Fisher Scientific, Rockford, IL, USA) for VAChT and α7-nAChR assay. Equivalent
amounts of protein (50 μg) were subjected to SDS-PAGE and then transferred to a
polyvinylidene fluoride membrane. After blocking with 5% BSA at room temperature for 2 h,
the membrane was incubated with specific primary antibodies against VAChT (1:1,000),
7-nAChR (1:3,000), or p-VEGFR2 (1:1,000), at 4°C overnight. After incubation with
peroxidase-conjugated second antibody (1:50,000) for 1 hour, enhanced chemiluminescence
was carried out (Thermo Fisher Scientific, Rockford, IL, USA). The blots were stripped and
re-probed with an antibody against -actin (1:40,000).
Tube Formation Assay for CMECs
The ECM gel was thawed at 4°C and brought to homogeneity using a cooled pipette. The
bottom of the cell culture plate (96-well) was coated with a thin layer of ECM gel (50 μl/well;
polymerization time: 60 min at 37°C). CMECs (3×104) in 150 μl DMEM was added on
solidified ECM gel and incubated for 6 h at 37°C. Endothelial tube formation was observed
under a phase-contrast microscope. Average capillary tube branch points were counted in six
random view fields per well. The results are expressed as mean folds of branching compared
with the control group.
Figures
 7-nAChR
knockout or
SD rats
wild-type mice
(ketanserin,
VAChT,
0.6 mg/kg/d in
 7-nAChR
food or not)
2w
SAD in SD rats
1w
(pyridostigmine,
30 mg/kg/d in
Regional
2w
Myocardial
infarction
water or not)
blood flow
1m
2w
3d
SAD or sham
2d
operation
Vascular endothelial
2w
growth factor
in SD rats
2w
SBP, DBP, HR,
BRS, ∆ HR (induced
Capillary
by atropine sulfate,
density
0.03 mg/kg, iv)
Figure 1S. A flow chart for animal experiments on angiogenesis. BRS, baroreflex sensitivity;
DBP, diastolic blood pressure; HR, heart rate; SAD, sinoaortic denervation; SBP, systolic
blood pressure; SD, Sprague-Dawley.
120
60
0
450
HR (beats/min)
120
DBP (mmHg)
SBP (mmHg)
180
80
40
0
Sham
SAD
300
150
0
Sham
SAD
Sham
SAD
Figure 2S. SAD caused no change in SBP, DBP and HR in rats (n=30). Sham, sham
operation for SAD.
CD34
DiI AcLDL
CD34＋DiI AcLDL
Figure 3S. Identification of primary cultured CMECs. Confluent CMECs were incubated
with 10 μg/ml DiI AcLDL overnight. Cells were fixed with 2% paraformaldehyde, incubated
with an antibody against CD34 for 1 hour, and incubated with FITC-labeled anti-goat IgG for
30 minutes. Cells positive for both DiI AcLDL and CD34 were judged to be CMECs. The
culture contained >95% CMECs. Scale bar=500 μm.