Methods, Supplemental
Lung Histology
Paraformaldehyde-fixed lungs were embedded in paraffin and hematoxylin and eosin
staining was performed on five μm sections. The first ten blood vessels (on end), with
diameters of 20 to 50μm, were used for analysis. The pulmonary vessel wall thickness-to-blood
vessel wall diameter (WT/D) ratio was determined by measuring the thickness of the vessel wall
(internal lamina to adventitia) divided by the intraluminal diameter (internal lamina to internal
lamina). The degree of pulmonary blood vessel wall muscularization was assessed and
reported as follows: 0: no smooth muscle, +: < ¼ muscularized, ++: < ½ muscularized, +++: >
½ muscularized, ++++: fully muscularized.
Measurement of RV hypertrophy
Cardiac atria were removed and the RV free wall was weighed. Wet weight
measurements were normalized to body weight (mg/g) and expressed as the RV free wall
weight-to-body weight (RV/BW) ratio.
EV Quantification
EV were resuspended in PBS and quantified using a laser-illuminated microscopic
technique with the NanoSight NS500 (NanoSight, UK). EP Brownian motion was tracked for
30-60 seconds and recorded. Over 500 tracks were recorded for each sample and the average
track length was used to estimate EV size. EV concentration was recorded and normalized to
the volume of blood collected (PEV) or to the number of whole lungs or whole hearts used for
EV. EV size range, mean and mode size were also recorded.
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Proteomic analysis of EV
Total protein isolated from EV was quantified using the BCA Protein Assay Kit (Pierce).
Protein samples were run on a NuPAGE® 4-12% Bis-Tris precast Gel (Invitrogen) and stained
with SimplyBlue® SafeStain (Invitrogen). The sample lane from the destained gel was cut into
ten equal gel pieces for fractionation. Each piece was washed with 50% acetonitrile/50% HPLC
grade water, reduced with DTT and alkylated with iodoacetamide. Pieces were digested with
modified trypsin (Promega) and the resulting peptide mixtures from each gel piece were
analyzed separately by data-dependent microcapillary reversed phase liquid chromatography
tandem mass spectrometry (LC/MS/MS). LC/MS/MS was performed using an Easy-nLC
nanoflow HPLC (Thermo Fisher) with a self-packed 75 µm idx15 cm C18 column coupled to a
LTQ-Orbitrap XL mass spectrometer (Thermo Fisher) in the data dependent acquisition and
positive ion mode (300 nL/min). Generated MS/MS spectra were searched against the nonredundant Murine IPI database by using Mascot (Matrix Science). Protein quantification was
achieved by employing MSQuant software (http://msquant.alwaysdata.net/).
Real-time RT-PCR analysis
10ng of RNA/sample was used to amplify cDNA using the High Capacity cDNA
transcription kit (Applied Biosystems, all equipment/reagents). RT-PCR reactions were
performed 7900HT Fast RT PCR System using 384-well TaqMan Array Cards preloaded with
the following primers: β2 microglobulin(Mm00437762_m1), E-selectin(Mm00441278_m1),
Endoglin (Mm00468256_m1), VE cadherin(Mm03053719_s1), CD143(Mm00802048_m1),
CD31(Mm01242584_m1), CD133(Mm00477115_m1), CD34(Mm00519283_m1), ckit(Mm00445212_m1), CXCR4(Mm01292123_m1), TNF RI(Mm00441875_m1), Erythropoietin
R(Mm00833882_m1), IL-6(Mm00446190_m1), IL-1 RI(Mm00434237_m1), VEGF
R1(Mm00438980_m1), VEGF R3(Mm01292604_m1), PDGF(Mm01205760_m1), endothelin1(Mm00438656_m1), 5-HTT(Mm00439391_m1), prostaglandin D2
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synthase(Mm01330613_m1), BMP receptor, type II(Mm00432134_m1),
eNOS(Mm00435217_m1) and VEGF R2(Mm01222421_m1). The 2-ΔΔCT method was used to
calculate relative expression of each target gene26. Additionally, microRNA microarray analysis
was performed using TaqMan® Rodent MicroRNA Array Cards.
Tracking of PKH26-labeled MCT-EV after infusion into normal mice
Vehicle-LEV,- PEV and MCT-LEV, -PEV were labeled with PKH26 dye (Sigma), per
manufacturer’s instructions. 100µl of each EV population or PBS (control) was injected by tail
vein into cohorts of normal C57BL/6 mice. 30 minutes later, mice were sacrificed. Lung frozen
sections were counterstained with the nuclear label DAPI and analyzed using conventional and
deconvolution fluorescence microscopy (Zeiss Axioplan 2 microscope, Carl Zeiss). PKH26+ EV
adjacent to pulmonary vascular endothelial cells were identified. Sections were photographed at
63x magnification using the AxioVision software package (Carl Zeiss). No photosubtraction or
processing of the artifact was performed.
Tracking of PKH26-labeled EV after incubation with BMPCs
BMPCs (Lin- cells, 107/culture well) were incubated in DMEM low glucose media with 1.5
x107 PKH26-labeled vehicle-LEV or -PEV, MCT-LEV or -PEV or an equal volume of PBS
(control) for two days at 37°C. Culture wells were imaged using fluorescence and light
microscopy. Cells were then removed from culture, washed with 50ml of PBS and centrifuged
at 300g for 10 min. The supernatant was discarded and cells were resuspended in 50ml of PBS
and centrifuged again at 300g for 10 min. Cells were then resuspended in 100ul of PBS and
imaged using fluorescence and light microscopy.
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Figure Legend, Supplemental Figures
Supplemental Figure 1. mRNA content of EV and gene expression profile of lineagedepleted (Lin-) bone marrow cells incubated with EV from vehicle-treated and MCTinjured mice. (A) Relative levels of endothelial progenitor cell (EPC) and endothelial cell
mRNA species and mRNA species known to be abnormal in PAH in MCT-PEV vs. vehicle-PEV,
MCT-LEV vs. vehicle-LEV. +++ mRNA present in MCT-EV but absent in vehicle-EV. (B)
Relative expression of EPC genes, endothelial cell genes and genes known to be abnormally
expressed in PAH in Lin- cells incubated with MCT-PEV vs. expression in Lin- cells incubated
with vehicle-PEV, Lin- cells incubated with MCT-LEV vs. expression in Lin- cells incubated with
vehicle-LEV.
Supplemental Figure 2. EV-based proteins specific to various cell types. (A) Vehicle,
MCT-PEV, (B) Vehicle, MCT-LEV based proteins specific to various cell types, expressed as a
percentage of all EV-based proteins of an identifiable cell source.
Supplemental Figure 3. PKH26-labeled MCT-PEV in the pulmonary vasculature of a
normal mouse. (A) PKH26 (red)-labeled MCT-EVs adjacent to a pulmonary vascular
endothelial cell (blue, nuclear counterstain DAPI), DAPI/Rhodamine/FITC filters. (B) Magnified
image of cell in red dashed box. Red bar = 10µm.
Supplemental Figure 4. Gene expression of bone marrow cells and mRNA content of
circulating EV from mice infused with EV. (A) Relative levels of EPC and endothelial cell
mRNA species and mRNA species known to be abnormal in PAH in PEV isolated from mice 14,
28 and 42 days after infusion with MCT-PEV vs. PEV from mice infused with vehicle-PEV, PEV
isolated from mice infused with MCT-LEV vs. PEV from mice infused with vehicle-LEV. +++
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mRNA present in PEV from mice infused with MCT-EV but absent in PEV from infused with
vehicle-EV. (B) Relative expression of EPC genes, endothelial cell genes and genes known to
be abnormally expressed in PAH in WBM isolated from mice 14, 28 and 42 days after infusion
with MCT-PEV vs. WBM expression in mice infused with vehicle-PEV, WBM isolated from mice
infused with MCT-LEV vs. WBM expression in mice infused with vehicle-LEV.
Supplemental Figure 5. PKH26-labeled EV are not present in outside of BPMC cells at
the time of cell transplantation. Lineage-depleted bone marrow cells incubated with PKH26labeled MCT-LEV. Cells were imaged in their culture well prior to cell harvest. (A) light, (B)
rhodamine filter, (C) both filters. EV are present within cells (white arrows) or in the culture
media unassociated with cells (white asterix). Cells were then washed with PBS, resuspended
in PBS and imaged. (D) light, (E) rhodamine filter, (F) both filters. EV are only present within
cells (white arrows). No EV are visualized outside of cells. Red bar = 50µm.
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Supplemental Table 1. Concentration mean size, size range of all EV populations
Concentration
Mean Size
Size Range
(x107, per ml blood
or set of lungs, +SD)
(nm, +SD)
(nm)
Control LEV
58.3 (6.9)
114.9 (45.7)
38-423
MCT LEV
55.7 (5.7)
105.9 (50.5)
42-402
Control PEV
17.6 (2.3)
82.5 (34.2)
31-289
MCT PEV
19.2 (3.4)
88.1 (43.3)
30-356
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Supplemental Table 2. Proteins characteristic of exosomes and microvesicles
that were isolated from all EV populations studied
Exosome Proteins
Microvesicle Proteins
Tetraspanins
Membrane Proteins
CD9, CD81
CD40 Lingand, Platelet glycoprotein
Ib, β1 integrin, P-selectin
Chaperone Proteins
Heat Shock proteins 70, 90
Membrane Trafficking Proteins
Ras-related proteins Rab-18, -27b,
Ras-related protein Rap-1b
Multivesicular Body Formation Proteins
Alix, Clatherin
Cytoskeletal Proteins
Myosins-1, -1d, -3, -4, -6; Cofflin
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Reference
1. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time
quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001;25:402-408.
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