Download Data Supplement Hypertension

Data Supplement
Expression and Function of Soluble Guanylate Cyclase in Pulmonary Arterial
Hypertension
Ralph Theo Schermuly, PhD; Johannes-Peter Stasch, PhD #; Soni Savai Pullamsetti, PhD;
Ralf Middendorff, MD *; Dieter Müller, PhD *; Klaus-Dieter Schlüter, MD §; Alexandra
Dingendorf §; Sascha Hackemack; Ewa Kolosionek; Christina Kaulen, Rio Dumitrascu, MD,
PhD; Norbert Weissmann, PhD; Joachim Mittendorf, PhD #; Walter Klepetko, MD &; Werner
Seeger, MD; Hossein Ardeschir Ghofrani, MD; Friedrich Grimminger, MD, PhD.
sGC activation in pulmonary hypertension
University of Giessen Lung Centre (UGLC), Giessen, Germany.
*
Department of Anatomy and Cell Biology, Giessen, Germany.
#
Pharma Research Center, Bayer HealthCare, Wuppertal, Germany.
&
§
Department of Cardiothoracic Surgery, University of Vienna, Vienna, Austria.
Institute of Physiology, Giessen, Germany
Corresponding author:
Ralph Schermuly, University of Giessen Lung Centre (UGLC), Klinikstrasse 36, 35392
Giessen, Germany, Phone ++49 641 994-2420, Fax ++49 641-994-2419.
Email: [email protected]
Materials and Methods
Immunoblot analyses
Frozen (liquid nitrogen) human lung tissue samples (~ 100 mg each), enwrapped in tin
foil, were pulverized by strokes with a hammer and homogenized at 4°C in 1 ml
homogenization buffer (50 mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 1 mM
dithiotreitol, and 0.1 mM phenylmethylsulfonylfluoride) by 10 to 15 strokes in a PotterElvehjem homogenizer. After centrifugation at 4,000 x g for 6 min at 4°C to remove cell
debris and nuclei, the supernatant fractions were re-centrifuged at 4°C for 30 min at 100,000 x
g. The resulting supernatants were used as soluble protein fractions. Protein concentrations
were determined (Bradford assay; Bio-Rad Laboratories, Munich, Germany) with BSA
(fraction V) as standard.
After separation by SDS-PAGE under reducing conditions in 8% acrylamide gels,
proteins were transferred to nitrocellulose membranes. After blocking, blots were probed with
an antibody directed against sGC (diluted 1:5,000, Alexis, San Diego, CA, USA). Anti-rabbit
IgG, linked to peroxidase (Pierce, Rockford, IL, USA), served as secondary antibody. Signals
were detected using enhanced chemiluminescence (Amersham) on Fuji X-ray films.
Reverse transcription-polymerase chain reaction (RT-PCR)
Specific primers used for amplification were: human sGC1, 5'- CAG TCG GAG CCG AGT
CTA TC -3' (forward) and 5'- TGC TTT GCC AAT GTT CTC TG -3' (reverse); human
sGC2, 5'- GCC TCA GAC GAT ACA GCA GA -3' (forward) and 5'- TTG GAG TGG TCT
GCA TAG GA -3' (reverse); human sGC1, 5'- GAG GTG TGG GAA GAC ATC AAA -3'
(forward) and 5'- GCA GCA GCA ACC AAA TCA TA -3' (reverse); human sGC2, 5'- GCT
TTG CCA ATG TTC TCT GA -3' (forward) and 5'- GTC GGA GCC GAG TCT ATC TT -3'
(reverse); human PBGD, 5'- CCC ACG CGA ATC ACT CTC AT -3' (forward) and 5'- TGT
CTG GTA ACG GCA ATG CG -3' (reverse); rat sGC1, 5'- GTC ACC CCC AAA GAA
AGA CA -3' (forward) and 5'- TGC TTT GCC AAT GTT CTC TG -3' (reverse); rat sGC2,
5'- TTG ATC ACC AGT GTG GCT TT -3' (forward) and 5'- AAT GGG TCT TCC ATC
AGG AG -3' (reverse); rat sGC1, 5'- TGA TAC CAT CTT GCG TGT CC -3' (forward) and
5'- AAT GAG CCC TTT GCC TTT TT -3' (reverse); rat sGC2, 5'- CGG ACA CTT GGA
GGA AAT CT -3' (forward) and 5'- CTC CTT CCT CCA CTC GAA AG -3' (reverse); rat
PBGD, 5'- CAA GGT TTT CAG CAT CGC -3' (forward) and 5'- ATG TCC GGT AAC GGC
GGC -3' (reverse).
Pharmacological profile of BAY 63-2521
We purifed rat sGC by using a baculovirus/Sf9 expression system and measured enzyme
activity as described (1). Briefy, sGC (0.16 mg protein/ml) was incubated for 10 min at 378C
in a final volume of incubation buffer ((mM): TEA/HCl 50, EGTA 100, IBMX 1,
dithiothreitol 1, cyclic GMP 1, creatine phosphate 5, 12.5 U/ml creatine phosphokinase,
MgCl2 3, 1 mg/ml BSA and 0.1 mCi [a-32P] GTP; pH 7.5) in the presence and absence of
sGC activator. Incubations were stopped by coprecipitation of 5'-nucleotides with a 400 ml
zinc acetate (100 mM) and 500 ml sodium carbonate (120 mM) solution. Following
centrifugation (5 min, 2800 g, 48C), [a-32P]cGMP was isolated from the supernatant by
chromatography on neutral aluminia colums. The amount of [a-32P]cGMP was determined by
liquid scintillation counting. All measurements were performed in duplicate and were
repeated at least three times. Haem-free sGC was prepared by low concentrations (0.5%) of
the non-ionic detergent Tween-20 without destruction of basal activity (1).
The preparation of PDE isoenzymes and inhibition of PDE enzyme activity have been
previously described (2).
Animals
Adult male C57Bl/6J mice and Spraque-Dawley rats (300-350 g body weight) were obtained
from Charles River Laboratories. Animals were housed under controlled temperature (≈22
°C) and lighting (12/12-hour light/dark cycle), with free access to food and water. All
experiments were performed according to the institutional guidelines that comply with
national and international regulations.
Exposure to Chronic Hypoxia
The animals were exposed to chronic hypoxia (10% O2) in a ventilated chamber, as described
previously (3). The level of hypoxia was held constant by an auto regulatory control unit
(model 4010, O2 controller, Labotect; Göttingen, Germany) supplying either nitrogen or
oxygen. Excess humidity in the recirculating system was prevented by condensation in a
cooling system. CO2 was continuously removed by soda lime. Cages were opened once a day
for cleaning as well as for food and water supply. The chamber temperature was maintained at
22–24°C. Control normoxic mice were kept in identical chambers under normoxic condition.
Hemodynamic measurements
Mice or rats were anaesthetised with ketamine/xylazine (i.p.) and placed on a heating pad to
maintain the body temperature. Artificial ventilation was performed through tracheostomy,
with 10 ml per kg bodyweight (BW) and a frequency of 120 respectively 90 breaths per
minute (SAR830A/P, IITC, Woodland Hills, CA). As previously described, systemic arterial
pressure (SAP) was monitored by catheterizing the left carotid artery with a polyethylene
cannula connected to a fluid-filled force transducer (Braun, Melsungen, Germany). The right
jugular vein was used for catheterisation of the right ventricle with a custom-made silicone
catheter to measure right ventricular systolic pressure (RVSP). The transducers were
calibrated before every measurement.
Radiotelemetric measurements of systemic arterial pressure, right ventricular pressure
and heart rate
The system (Dataquest A.R.T. 2.1; Data Science Inc) comprises a fluid –filled sensing
catheter (5 cm long, external diameter 0.7 mm, internal diameter 0.25 mm; model TA11PA)
connected to a transmitter that signals to a remote receiver (model RPC-1) and a data
exchange matrix connected to a computer. For continuous measurement of right ventricular
pressure (RVSP), systemic arterial pressure (SAP) and heart rate, radio telemetric sensors
were implanted into anaesthetised mice via a closed chest technique. After surgery, mice were
allowed to recover for 3 days. None of the animals manifested signs of inflammation or
infection. After recovery the animals for long term investigation were placed in hypoxic
conditions and treated with sGC stimulator Bay63-2521 10mg/kg body weight from day 21 to
day 35 as described above. For the acute effects of Bay 63-2521, SAP was investigated.
Different doses of Bay 63-2521 were orally applied and SAP online recorded for the next 48
hours.
Isolated Perfused Mouse Lung
Lungs from mice exposed chronically to normoxia or hypoxia for three weeks were
investigated. The effect of soluble guanylate cyclase (sGC) stimulation on acute hypoxic
pulmonary vasoconstriction (HPV) was examined in isolated ventilated perfused mice lungs
(4) from animals which were kept under normoxic or hypoxic conditions. The technique of
successive hypoxic maneuvers in buffer-perfused lungs has been previously described (5).
Sequential hypoxic maneuvers of 10-min duration interrupted by 15-min periods of normoxia
were performed. The effect of Bay63-2521 on pressure responses provoked by alveolar
hypoxia (1 % O2) was determined within such a sequence of repetitive hypoxic maneuvers.
The agent was added to the buffer fluid 5 min before a hypoxic challenge, the addition
starting after the second hypoxic maneuver was accomplished. Cumulative dose-effect curves
were established by addition of Bay63-2521 (dose range: 0.01 – 1.0 µM).
Tissue Processing
After recording systemic arterial and right ventricular pressure, carotid artery was sectioned
and blood was collected for hematocrit measurement and plasma separation. The lungs were
first flushed with saline solution and than perfused with paraformaldehyde solution through
pulmonary artery and saline through trachea with a constant pressure of 22 and 11 cm H2O
respectively. Lung and heart was removed en block. The lungs were further stored in Zamboni
fixative and phosphate buffer till paraffin embedding and hearts were used for right heart
hypertrophy assessment. The RV was dissected from the left ventricle + septum (LV + S) and
these dissected samples were dried and weighed to obtain the right to left ventricle plus
septum ratio (RV/(LV+S)).
Morphometric analysis
As described previously, the lung lobes were embedded in paraffin blocks and sections of 3
µm were cut (6). The degree of muscularization of small peripheral pulmonary arteries was
assessed by double-staining the sections with an anti--smooth muscle actin antibody
(dilution 1:900, clone 1A4, Sigma, Saint Louis, MO) and anti-human von Willebrand factor
antibody (vWF, dilution 1:900, Dako, Hamburg, Germany). Sections were counterstained
with methyl green and examined by light microscopy using a computerized morphometric
system (Qwin, Leica, Wetzlar, Germany). At 40× magnification, 80–100 intraacinar vessels
accompanying either alveolar ducts or alveoli were analyzed by an observer blinded to
treatment in each mouse. Each vessel was categorized as non-muscularized, partially
muscularized or fully muscularized (7;8). The percentage of pulmonary vessels in each
muscularization category was determined by dividing the number of vessels in that category
by the total number counted in the same experimental group.
Figure 1. Chemical Structure of BAY 63-2521
Figure 2. sGCα1 expression in lungs from healthy and IPAH patients.
smooth muscle actin staining is demonstrated in the upper row for identifying vascular
smooth muscle cells. sGCα1 expression in human lungs from healthy and IPAH patients is
demonstrated in the lower row by positive staining in the medial layer of small pulmonary
arteries.
Figure 3. Dose-response curve of BAY 63-2521 on acute hypoxic pulmonary
vasoconstriction in isolated mouse lungs.
In a sequence of repetitive hypoxic challenges (1 % O2, 10 min), alternating with normoxic
ventilation periods (21 % O2, 15 min), cumulative doses of BAY 63-25221 were applied 5
min after cessation of each hypoxic challenge in lungs from animals which were kept under
normoxic (circles) or hypoxic (triangles) conditions for 21 days. Data are referenced to the
initial hypoxic response (=100%).
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