Materials and Methods
Animals. Male C57BL/6 wild type, TSP1- and CD47-null mice (stock numbers 000664, 006141
and 003173 respectively) were obtained from the Jackson Laboratory (Bar Harbor, ME) and
housed for one week prior to use in experiments. Mice had ad libitum access to water and
standard rodent chow at all times. Sprague Dawley rats were purchased from Harlan
(Indianapolis, Ind.). All studies were performed using protocols approved by the Institutional
Animal Care and Use Committee (IACUUC) at the University of Pittsburgh or using protocols
approved by the Committee for Research and Ethics of the Universidad Autonoma de Madrid and
in accordance with Directive 2010/63/EU of the European Parliament. Adequacy of anesthesia
was monitored at all times by assessment of skeletal muscle tone, respiration rate and rhythm and
response to tail pinch. Euthanasia was achieved by isoflurane inhalational anesthesia (1.5%) and
concurrent cervical dislocation as approved by the respective University IACUCs.
Cell cultures. Primary murine pulmonary microvascular endothelial cells were harvested from 810 week old male C57BL/6 wild type, TSP1- and CD47-null mice. Following humane euthanasia,
murine lungs were harvested sterilely, placed in growth medium and cut into 2-5 mm pieces.
Tissue fragments were then incubated in 0.5% collagenase (Worthington) at 37 ºC for 30 m with
intermittent trituration. The resultant was then transferred to sterile 25 cm2 culture flasks in full
endothelial growth medium. On reaching confluence cells were harvested and selection for
endothelial cells performed with CD31-coated ferrous beads. Human pulmonary arterial
endothelial cells were purchased from Lonza and maintained in the recommended proprietary
medium.
Human subjects. After obtaining informed consent, whole human lung tissue was obtained from
patients at the time of lung transplant and rapidly frozen in liquid nitrogen (John Hopkins
University – PAH patients and the University of Pittsburgh – non-PAH controls) under ongoing
IRB approval from respective institutions and in accordance with the principles outlined in the
Declaration of Helsinki.
Murine chronic hypoxic exposure model of PAH. Male 9-12 wk old wild type, TSP1- and
CD47-null mice were placed in an airtight Plexiglass chamber with a ProOx Model 110 Oxygen
Controller (Biospherix Ltd.) and exposed to chronic hypoxia (FiO2=0.10) for 21days under
normobaric conditions. Mice from both strains maintained in room air served as normoxic
controls (n=8 for all groups). In acute hypoxia treatment experiments male 9-12 wk old wild type
and TSP1 null mice were exposed either to 21% oxygen levels (Nx) or placed in an Invivo2 400
Hypoxia Work Station (Ruskinn Technology) and exposed to 13% oxygen for 1 h and then 7.5%
oxygen levels (Hx) for the indicated time points. Afterward animals were sacrificed and lung
tissue samples were processed for RNA isolation and protein extracts. TSP1 protein levels were
analyzed by western blot and mRNA changes induced by hypoxia versus normoxia were
analyzed by Real Time RT-PCR with the StepOne Plus (Applied Biosystems).
Hemodynamic and right ventricular measurements. Animals were anesthetized with
pentobarbital (40 mg/kg i.p.). The trachea was cannulated, and animals were ventilated with 1.5%
isoflurane. The thoracix cavity was opened, a Milar a catheter was inserted in to the right and left
ventricles and pressures measured using a PowerLab data acquisition system (AD Instruments).
For ventricular weight measurements, hearts were excised and atria were removed. The right
ventricle (RV) free wall was dissected, and each chamber was weighed. The ratio of RV weight
to left ventricular (LV) weight plus septum (RV/LV+S, Fulton Index) was used as an index of RV
hypertrophy.
Rat monocrotaline model of PAH. Monocrotaline sodium (mct) was administered as a single
50 mg/kg subcutaneous injection to male Sprague-Dawley rats weighing between 250 and 260 g.
Animals were treated with monocrotaline or monocrotaline plus a CD47 monoclonal antibody
(clone OX 101, Santa Cruz Biotechnology, 1 µg/gram weight i.p. on day 1 and day 14). Sham
rats received vehicle (normal saline) only. Right ventricular systolic pressure and Fulton Index
measurements were obtained 30 days after monocrotaline treatment following essentially the
same protocol as described above in mice. Lung sections were stained with hematoxylin and
eosin. Images for arterioles were captured with a microscope digital camera system (Provus,
Olympus), and arterial area was measured using an image analysis program (Metamorph,
Universal Imaging Corp.). Percent wall thickness was calculated by the following formula: wall
thickness (%) = area external − area internal/area external × 100, where area external and area
internal are the areas bounded by external elastic lamina and the lumen, respectively. Eight
vessels per rat of comparable size from the lungs of four different rats from both control, mct, and
mct + CD47 antibody groups were determined.
Lung histology. After hemodynamic measurements of the heart, mouse lungs were perfused
with PBS, fixed with 4% paraformaldehyde (PFA) and excised (12h PFA 4°C). Lungs were
transferred into 30% sucrose (over night at 4°C), paraffin embedded and sectioned (10µm).
Pulmonary vascular remodeling. Pulmonary vascular remodeling was assessed in lung sections
stained for α-smooth muscle actin (Sigma) by counting the number of partially and fully
muscularized peripheral arterioles (35-100 mm) per high power field (200x total magnification).
For each mouse, at least 20 high power fields were analyzed in multiple lung sections. Reviewers
were blinded as to strain and treatment.
Immunoprecipitation. Cells were plated to 80% confluency and serum starved for 24 hours and
then placed in medium containing 0.1% BSA for one hour prior to the experiment. CD47
blocking antibody (1 µg/ml, clone B6H12, Santa Cruz) was added 30 minutes after the addition
of serum free medium and TSP1 was added 1 hour after the addition of serum-free medium. Cells
were placed in a hypoxia chamber at an oxygen tension of 1% for 3 hours. Cells were washed
twice with ice-cold PBS, scraped in NP-40 lysis buffer containing 1mM sodium fluoride, 1mM
sodium orthovanadate, 1x phosSTOP (Roche) and protease inhibitor cocktail (Sigma) and
collected. Cells were rotated at 4º C for 30 minutes and centrifuged for 20 minutes at 13000 rpm.
The supernatants were transferred to a new tube and protein concentration assessed using the DC
Protein Assay (BioRad). One hundred micrograms of protein was added to 0.5 µg total CD47,
caveolin-1 antibody, or IgG isotype control (all Santa Cruz) and rotated overnight at 4º C. Protein
G agarose beads (10 µL, Pierce) were added and rotated for 2 hours at room temperature. IP
buffer was added and samples centrifuged at 2500 g for 3 minutes. The supernatants were
discarded and the process repeated three times. De-ionized water was added for the final wash.
Samples were then suspended in 2x Laemmli buffer (BioRad), boiled for 5 minutes at 95º C,
placed on ice for 2 minutes and pulsed before evaluation by SDS-Page.
Protein expression by Western blot analysis. Lysates of lung tissues (human, murine and rat)
or cells were prepared and subjected to Western analysis. Thirty micrograms of total protein was
boiled, resolved by SDS-page and transferred onto nitrocellulose (BioRad). In blots for CD47
non-reducing conditions were used with 6% SDS-PAGE. Blots were probed with primary
antibody to the respective proteins and were visualized after 1h incubation in secondary antibody
on an Odyssey Imaging System (Licor). The following antibodies were employed - rabbit
anti‐Caveolin‐1, Santa Cruz, 1:5000, Cat.No. sc‐894; mouse anti‐phospho‐eNOS, BD Bioscience,
1:1000, Cat.No. 612392; mouse anti‐eNOS, Invitrogen, 1:1000, Cat.No. 33‐4600; rabbit
anti‐eNOS, Santa Cruz, 1:1000, Cat.No. sc‐654; mouse anti‐thrombospondin, Abcam, 1:1000,
Cat.No. ab1823; anti-CD47 MAIP301: sc12731, rabbit anti-phospho-caveolin (Tyr14), Cat.No.
3251 and rabbit anti‐β‐actin, Cell Signaling, 1:1000, Cat. No. 4967. The intensity of the bands
was quantified using the Odyssey software or Image J (rsbweb.nih.gov/ij/).
Determination of mRNA transcript. TSP1 mRNA expression levels in lung samples from wild
type and TSP1 null mice exposed to normoxia or hypoxia (normobaric 7.5% O2) for the indicated
time points was determined by QPCR. The following primers pairs were used: TSP1 forward: 5’
TGC GCA CCC TGT GGC ATG ACC 3’, TSP1 reverse: 5’ TCT TTG GCC TGT GGC TGA
GAC 3’. Representative data from 3 independent experiments are presented. mRNA levels are
expressed as fold change over normoxic conditions and normalized with HPRT levels as
endogenous control. TSP1 and CD47 mRNA expression levels in human lung samples were
determined using specific Taqman primers and probes for 18s (Hs_99999901_s1), CD47
(Hs00179953_m1) and TSP1 (Hs_00962908_m1) obtained from Applied Biosystems (Carlsbad,
CA). Total RNA was extracted using Qiagen RNeasy® Mini Kits (Qiagen, Hilden, Germany) as
per the manufacturer instructions. RNA was quantified using the Take3 Gen5 spectrophotometer
(BioTek, Winooski, VT). One microgram (1µg) of RNA was treated with DNase I (amplification
grade, Invitrogen, Grand Island NY) and then reverse-transcribed using the Superscript III First
Strand Synthesis Supermix (Invitrogen). cDNA was amplified using Platinum® Quantitative
PCR SuperMix-UDG (Invitrogen) in 20 µl volumes in triplicate in a 384 well plate with gene
specific primers and probe on the ABI Prism 7900HT Sequence Detection System (Applied
Biosystems), according to manufacturer’s instructions. Thermal cycling conditions were 50ºC for
2 minutes, 95ºC for 2 minutes, followed by 40 cycles of 95ºC for 15 seconds and 60ºC for 1
minute. Data were analyzed using the ΔΔCt method with expression normalized to the
housekeeping gene (18s) and subsequently to normal human lung tissue.
Determination of monomer to dimer ratio of eNOS protein. Tissue samples were prepared as
described above for Western analysis with the following exceptions: Samples of equal protein
content were kept for 5 min at room temperature and one sample was boiled to serve as control
for monomer formation. Samples were then resolved on an 8% SDS-Page on ice, at 100V and
transferred and blotted against total eNOS [mouse anti-eNOS (Invitrogen) for mouse samples,
rabbit anti-eNOS (Santa Cruz Biotechnology) for human samples]. Intensity of bands was
quantified as above.
Measurement of intracellular O2.-. hPAEC were grown to confluence and then placed in a
humidified chamber within a glove box and maintained at 37ºC, 5% CO2, 1% O2 for 24h.
Normoxic controls were maintained in a standard cell culture incubator at 37ºC, 5% CO2. DHE
(5µM) was added for the final 30 min of the 24h incubation period. O2·- was evaluated by
measuring the conversion of DHE to hydroxyethidium in a fluorimeter (TECAN Infinite M200)
using an excitation and emission wavelengths of 495nm and 567nm respectively.
siRNA knockdown of Cav-1. All siRNAs were designed and synthesized by Dharmacon
(ThermoFisher Scientific). siRNA transfection was performed using a lipofectamine reagent
(Invitrogen) according to the manufacturer’s instructions. Scrambled siRNAs were used as
negative controls. To achieve optimal transfection efficiency, transfection reagent, siRNA, cell
density, and time of transfection were optimized. Gene silencing was monitored by Western blot
analysis 72h after transfection.
Gain-of-function studies in pulmonary endothelial cells. Murine pulmonary endothelial cells
were harvested from age matched male wild type and TSP1 null mice and used at passage 1-3.
Cells were plated at 80% confluence, treated with normoxia or hypoxia (1% O2) ± exogenous
TSP1 (2.2 nmol/L) for 24 h, cell lysate prepared and Western blot analysis for Cav-1, CD47 and
α-tubulin expression performed.
Statistics. Statistics were performed using GraphPad Prism software. Data were analyzed by 1way ANOVA followed by Tukey test for multiple comparisons. For grouped analysis, data were
analyzed by 2-way ANOVA followed by Bonferroni post hoc test.