Supplementary Methods
Materials and Methods
Animals
Male Sprague Dawley rats (aged circa 4 months, 250-300g) were used in these studies and
were obtained from Charles River UK. Animals were treated in accordance with the Animals
(Scientific Procedures) act 1986 published by the UK Home Office and the Guide for the
Care and Use of Laboratory Animals published by the US National Institutes of Health
(Publication No. 85-23, revised 1996).
Primary cardiomyocyte isolation
Adult rats were anesthetized with i.p. Na-pentobarbital, the hearts were isolated and
perfused via the aorta. Cardiomyocytes were isolated by collagenase II perfusion using a
standard method and plated on laminin (1). Cells were subject to hypoxia and reoxygenation with a buffer simulating the ischemic milieu containing 128 mM NaCl, 2.2 mM
NaHCO3, 14.8 mM KCl, 1.2 mM MgSO4, 1.2 mM K2HPO4, 1 mM CaCl2, 10 mM Na-lactate
(pH 6.4) in a hypoxic chamber containing 95% N2/ 5% CO2 for 3 h, after which cells were
placed in a standard incubator in normal medium for 1 h. Control cells were incubated in
normoxic medium 118 mM NaCl, 22 mM NaHCO3, 2.6 mM KCl, 1.2 mM MgSO4, 1.2 mM
K2HPO4, 1 mM CaCl2, 10 mM glucose (pH 7.4 gassed with 95% O2 / 5% CO2).
Cell culture
HL-1 cells are a cardiac muscle cell line derived from the AT-1 mouse atrial cardiomyocyte
tumor lineage, which retain in vitro phenotypic characteristics of the adult cardiomyocyte (2).
They were cultured according to standard methods (2). HL-1 cells were subject to 18 h
hypoxia followed by 1.5 h reoxygenation according to the above method.
Rat exosome preparation
Rats were anesthetized with 25 mg/kg Na-pentobarbital i.p. and placed on a heated mat.
After thoracotomy, 4 ml blood was rapidly removed into a citrated vaccutainerTM to minimize
platelet activation. Exosomes were prepared by standard differential centrifugation as
follows: centrifugation at 1,600 x g 20 min 4 °C to obtain plasma, then 10,000 x g 30 min
4 °C to remove cells and platelets, then twice at 100,000 x g 60 min, 4 °C with a SW-41
rotor, washing with PBS. The vehicle control consisted of PBS alone.
Human exosome preparation
These experiments were conducted according to Declaration of Helsinki principles. All
human and animal studies were approved by the appropriate institutional review boards. The
study of human samples was performed according to ethics approval reference 13/LO/0222.
After written consent, exosomes were isolated from healthy, 30-45 year old, male volunteers,
after overnight fasting. 60 ml blood was drawn from the antecubital vein by butterfly syringe
into citrated vaccutainersTM (BD Biosciences). The first two vaccutainers were excluded from
the exosome isolation procedure to avoid artifacts due to platelet activation. Exosomes were
isolated by centrifugation at 1,600 x g 20 min 4 °C, then 10,000 x g 30 min 4 °C, then 3
times at 100,000 x g 60 min, 4 °C.
Rat and human exosome samples were tested for endotoxin contamination using the LAL
Chromogenic Endotoxin Quantitation Kit (Pierce). Endotoxin levels were <0.1 EU/ml, such
that when exosomes were added to the cells, endotoxin concentration was <0.001 EU/ml,
well below the minimum able to activate TLR4 (3).
Remote ischemic preconditioning
After taking control samples, blood was taken from the same individuals a second time, after
remote ischemic preconditioning (RIC), in order to ascertain if this phenomenon had any
effect on exosome production. In these subjects, the contralateral arm was subject to an RIC
protocol consisting of inflation of a pressure cuff to 200 mmHg for 5 min and releasing for 5
min, repeated for 3 cycles. Blood was drawn from the antecubital vein as above.
Electron microscopy
Electron Microscopy was performed on a Joel 1010 transmission electron microscope (Joel
Ltd, Warwickshire, UK) after a standard staining procedure with 0.5% uranyl acetate (4).
Immunostaining was performed on whole-mounted exosomes using cmHsp70.1 followed by
a gold-labelled goat anti-mouse IgG secondary antibody, followed by post-fixation and
staining (4).
Nanoparticle tracking analysis
Nanoparticle tracking analysis (NTA) is based on the principle that the rate of Brownian
movement of nanoparticles in solution is related to their size (5). In this method, a 405 nm
laser light is directed at a fixed angle to the vesicle suspension, and the scattered light is
captured using a microscope and high-sensitivity camera. By tracking the movement of
individual nanoparticles over time, the software can calculate their diameter. One advantage
of this method is the ability to measure size distribution in polydisperse samples. Exosome
preparations were examined using a Nanosight LM10-HS (Nanosight Ltd) as described (5)
with constant flow injection. Five recordings of 30 sec each were captured, analyzed and the
data from at least 5,000 individual particle tracks were analyzed per sample.
Incubations with chemical inhibitors and Western blot analyses
For survival experiments, exosomes were added to cells and incubated for 30 min. Where
indicated, inhibitors were added to cells 30 min before and throughout exosome treatment.
Inhibitors were used at the following concentrations: 10 μM wortmannin, 10 μM LY-294002,
50 μM PD-95059, 10 μM U0126, 10 μM SB203580, 5 μM TAK-242. Exosomes were
pretreated with antibodies (cmHsp70.1) for 30 min at a ratio of 1 μg antibody per 5 μg
exosomes. For signaling experiments, exosomes were added acutely for different times, as
indicated where pertinent, and the cells were rapidly lysed on ice (6). Primary antibodies
from BD Biosciences were used against CD81 (clone TAPA-1), and antibodies from Cell
Signaling Technologies: ERK1/2 (#9102), Phospho-ERK1/2 (Thr202/Tyr204)(#9101), Akt
(#9272),
Phospho-Akt
(Ser473)(#9271),
Hsp27
(total
Hsp27
and
phospho-
Ser15/78/82)(#12594), and Santa Cruz Biotechnology: beta-actin (#47778), tubulin (#5546),
Hsp70 (clone N27F3-4), CD63 (clone H193).
Flow cytometry
Exosome size (50-100 nm) does not allow direct analysis by flow cytometry, which has a
lower limit of detection of particles ~0.2 μm in diameter. To overcome this, a standard
protocol of exosome binding to aldehyde microspheres of 4 μm was used with modifications
(4). Briefly, macromolecular IgG complexes were cleared from 2 x 109 exosomes (quantified
by NTA) by an immunoprecipitation pre-clearing protocol using G protein sepharose (Protein
G sepharose, GE Healthcare). Exosomes were then bound to 5 μl of aldehyde/sulfate latex
microspheres (4% w/v, 4 µm Molecular Probes)(a ratio of ~330 exosomes per microsphere),
in 1 ml of sterile PBS with agitation at 4 ºC. Exosome-coated microspheres were blocked
with 3% fatty acid-free BSA for 1 h at 4 ºC, then incubated with isotype control or specific
primary antibodies: CD81 (clone TAPA-1), Hsp70 (clone N27F3-4), CD63 (clone H193), or
cmHsp70.1-FITC (7). Secondary antibodies Alexa Fluor488 or Alexa Fluor585 were used
when necessary. For measuring cell death of HL-1 cells, the mitochondrial potentiometric
cationic dye 3,3’dihexyloxacarbocyanine iodide (DiOC6(3)) was used to detect loss of
mitochondrial transmembrane potential (ΔΨm), in addition to the vital dye propidium iodide
(PI) as described previously (8). Fluorescence was detected simultaneously in each sample.
For the analysis of cells or microspheres, we used singlet-gated events on SSC/pulse-width.
All samples were analyzed using an Accuri C6 flow cytometer (BD Biosciences).
Langendorff isolated perfused rat heart model
Langendorff experiments were carried out as described previously (6). Rats were
anesthetized with sodium pentobarbital by intraperitoneal (i.p.) injection (50 mg/kg) and
adequacy of anesthesia was monitored during surgery by testing the pedal reflex. Hearts
underwent a stabilization period of 40 min followed by 35 min of regional ischemia and 120
min of reperfusion. Regional ischemia was induced by occlusion of the left anterior
descending coronary artery by tightening a 3.0 silk suture around the vessel. Vehicle (PBS)
or ~5 x 1010 exosomes (50 μg), from a single rat was added to the perfusate throughout the
final 15 min stabilization before ischemia. At the end of the experiment, the extent of
infarction was determined by fixing, slicing, and staining the heart with tetrazolium chloride
(6). The area of infarction is measured relative to the total ischemic area demarcated by
perfused with Evan’s blue. The experimenter performing the infarctions and measurements
was blinded to the treatment group.
In vivo rat experiments
Male Sprague Dawley rats were anesthetized with sodium pentobarbital (50 mg/kg i.p.). The
rats were intubated and ventilated with a Harvard ventilator (70 strokes/min, tidal volume: 810 ml/kg,). Body temperature was maintained at 37 ± 1°C by means of a rectal probe
thermometer attached to a temperature control system (CMA450). Once the animal was
stable, ~5 x 1010 exosomes (50 μg, i.e.: the entire exosome fraction from a donor rat), or
vehicle (sterile phosphate buffer solution), were administered by intravenous (i.v.) injection
as a 100 μl bolus over 10 s into the tail vein 15 min prior to thoracotomy. As a positive
control for cardioprotection, remote ischemic preconditioning was induced by inflating a
pressure cuff to 200 mm Hg around the right hind limb for 3 periods of 5 min separated by 5
min. Left lateral thoracotomy was performed to expose the heart and a 6-0 suture placed
around the left coronary artery. The suture was tightened using a loop system to create left
anterior descending (LAD) artery ligation and regional ischemia which was confirmed by ST
elevation on the ECG (LabChart, AD Instruments), and blanching of the myocardium.
Following 30 min of ischemia, the myocardium was reperfused for 120 min. At the end of
reperfusion, the heart was removed from the chest, all the blood was flushed out, the LAD
permanently occluded and the heart perfused with 0.5% Evans blue to delineate the area at
risk. Hearts were frozen at −20°C for several hours before infarct size determination by
staining with tetrazolium chloride and image J software.
Statistical analysis
Data is shown as mean ± SEM. Pairwise comparisons were made by Student’s t-test. Oneway ANOVA was followed by post-test analysis using the Tukey test for multiple
comparisons. Two-way ANOVA was carried out followed by Bonferroni correction to test for
significance when performing multiple comparisons between different groups. P<0.05 was
considered significant.
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