Materials and methods
Drugs and reagents: All chemicals and reagents were purchased
from Sigma, Thermo-Fisher, and VWR.
Animal models and experimental protocols: All animal
experiments were approved by the New York Medical College Animal Care
and Use Committee. Male and female C57BL/6J mice (18-32 g) were
purchased from the Jackson Laboratory and randomly divided into four
groups: normoxia (Nx), normoxia+Sugen5416 (Nx+SU), hypoxia (Hx), and
hypoxia+Sugen5416 (Hx+SU) groups. Mice in Nx group were placed in
normoxic (21% O2) environment. Hx group was placed in a
normobaric hypoxic chamber (10% O2) for 6 weeks. Mice
in Hx+SU group received subcutaneous injection of SU5416 (20 mg/kg) once
weekly during 3 weeks of Hx (10% O2). We also separated
each group into non-treatment (Nx, Hx, and Hx+SU) and drug treatment
(Nx+4091, Hx+4091 and Hx+SU+4091) groups. Mice in the drug treatment
groups received daily subcutaneous injection of a novel G6PD inhibitor,
N-[(3β,5α)-17-Oxoandrostan-3-yl]sulfamide (PDD4091; 1.5 mg
kg-1 day-1) (Hamilton et al., 2012),
for 3 weeks. To determine whether PDD4091 reduces PH in a dose-dependent
manner, mice were randomized to receive low (0.15 mg
kg-1 day-1), medium (1.5 mg
kg-1 day-1), or high (15 mg
kg-1 day-1) dose injection of
PDD4091. In addition, we also determined the role of G6PD in the
pathogenesis of SU+Hx+Nx-induced PH in rats. Wild-type (Sprague Daley;
SD; 460-800 gm) rats and rats expressing loss-of-function MediterraneanG6pd variant (G6PDS188F; 350-750 gm), which
were recently generated in our laboratory (Kitagawa et al., 2020; in
press), received one subcutaneous injection of SU (20 mg/kg) and then
exposed to 3 weeks of Hx (10% O2) followed by 5 weeks
of Nx (SU/Hx/Nx). Hx and Hx+SU mice and rats are pre-clinical models of
PH (Stenmark, Meyrick, Galie, Mooi & McMurtry, 2009). At the end of the
treatment period, hemodynamic measurements were performed, tissue
(lungs, hearts, and arteries) were harvested, and blood samples were
collected. Data analysis was performed in blinded fashion.
Hemodynamic measurements: Closed-chest cardiac catheterization
was performed using an MPVS Ultra Single Segment Pressure-Volume Unit
(Millar Instruments, US) in combination with a cardiac catheter.
Briefly, mice were anesthetized with isoflurane (induced at 3% and
maintained at 1.5%) and placed on a heated table. RV systolic pressure
(RVSP) and RV end-diastolic pressure (RVEDP) were measured by
catheterization of the RV via the right external jugular vein using
Millar Mikro-Tip catheter (Model SPR-671, tip size of 1.4F, Millar
Instruments, US). The catheter was then removed, and the jugular vein
was tied off. For hemodynamic measurements from LV, the right carotid
artery was dissected and a Millar Mikro-Tip conductance catheter (model
SPR-839, tip size of 1.4F, Millar Instruments, US) introduced into the
artery and advanced into the LV via the aortic valve. Once
steady-state hemodynamics were achieved, pressure-volume (P-V) loops
were recorded and analyzed using LabChart 8 software (ADInstruments,
US).
Hematocrit measurements and blood chemistry analysis: After
hemodynamic measurements were completed, blood was collected from the
cardiac chambers into a heparinized syringe. Heparinized blood was
placed in capillary tubes, and hematocrit (%) was calculated as the
length of the erythrocyte layer divided by the length of the entire
blood sample. Plasma was shipped to Antech Diagnostics (NC, USA), a GLP
facility, where blood tests were performed with routinely used clinical
laboratory diagnostic tools.
Assessment of right ventricular hypertrophy: Following the
cardiac catheterization, the animals were euthanized by cervical
dislocation and whole hearts were excised and RV free wall and LV
including ventricular septum (S) were separated and weighed
independently. Fulton’s index (RV/LV + S ratio) was calculated as an
index of RV hypertrophy.
Isolation of small intrapulmonary arteries (IPA) and IPA tone
measurements: Mice (25-30g) were sacrificed by cervical dislocation and
small intrapulmonary arteries (IPA) of 3rd order
(100–150 μm in diameter) were isolated from the lung, dissected free of
connective tissue, and placed in Krebs bicarbonate buffer solution (pH
7.4) containing the following (in mM): 118 NaCl, 4.7 KCl, 1.5
CaCl2 x2H2O, 25 NaHCO3,
1.1 MgSO4, 1.2 KH2PO4,
5.6 glucose, and 10 HEPES. Then the vessels were mounted on a wire
myograph (Danish Myo Technology A/S, Aarhus, Denmark) and bathed in
Krebs buffer solution at 37°C and an optimal passive tension of 3 mN.
After 30 min of incubation, the arterial viability and equilibration
were assessed by the stimulation of the vessels with repeated 10 min
exposures to KCl (60 mM; 60K). For registration of vascular ring
contractile activity and its following analysis, Chart 5.5.4 and
LabChart Reader 8.1.9 (ADInstruments, Inc.) software were used. Vascular
tension is presented as a percentage of the maximum steady-state
contraction level obtained to the exposure to 60K.
Quantitative real-time PCR: Real time RT-PCR technique was used
to analyze mRNA expression. Briefly, total RNA was extracted from lungs
using a Qiagen miRNEasy kit (Cat # 217004). The input RNA quality and
concentration were measured on the Synergy HT Take3 Microplate Reader
(BioTek, Winooski, VT) and cDNA was prepared using SuperScript IV. VILO
Master Mix (Cat # 11756500, Invitrogen) for mRNA. Quantitative PCR was
performed in duplicate using TaqManTM Fast Advanced Master Mix (Cat #
44-445-57) for mRNA using a Mx3000p Real-Time PCR System (Stratagene,
Santa Clara, CA). The primers for the QPCR were purchased from Thermo
Fisher Scientific/TaqMan. Results for mRNA expression was normalized to
internal control Tuba1a , and relative mRNA expression was
determined using the ΔCt method.
RNA-Seq analysis: After collecting lungs from pulmonary
normotensive and hypertensive mice, total RNA was isolated from tissue
using the Qiagen All Prep DNA/RNA/miRNA Universal kit according to
manufacturer’s instructions. RNA was quantified using the NanoDrop
(ThermoFisher) and quality was assessed using the Agilent Bioanalyzer
2100. RNA-seq library construction was performed using the TruSeq
Stranded Total RNA Preparation kit (Illumina) with 200 ng of RNA as
input according to the manufacturer’s instructions. Libraries were
sequenced on the HiSeq2500 with single-end reads of 100nt at the
University of Rochester Genomics Research Center. Single-end sequencing
was done at a depth of 10 million reads per replicate (N=3).
Quantitative analysis, including statistical analysis of differentially
expressed genes, was done with Cufflinks 2.0.2 and Cuffdiff2
(http://cufflinks.cbcb.umd.edu). The Benjamini-Hochberg method was
applied for multiple test correction (FDR<0.05).
Reduced Representation Bisulfite Sequencing (RRBS): Genomic DNA
was isolated from lungs using the Qiagen All Prep DNA/RNA/miRNA
Universal kit according to manufacturer’s instructions. DNA was
quantified using the NanoDrop (ThermoFisher) and Qubit Fluorometer
(ThermoFisher). Genomic DNA quality was assessed using the Agilent
TapeStation. RRBS library construction was performed with the Premium
RRBS Kit (Diagenode) following the manufacturer’s instructions.
Libraries were sequenced on the HiSeq2500 with paired-end reads of
125nt. Raw reads generated from the Illumina HiSeq2500 sequencer were
demultiplexed using bcl2fastq version 2.19.0. Quality filtering and
adapter removal are performed using Trim Galore version 0.4.4_dev with
the following parameters: ” –paired –clip_R1 3 – clip_R2 3
–three_prime_clip_R1 2 –three_prime_clip_R2 2”
(http://www.bioinformatics.babraham.ac.uk/projects/
trim_galore/). Processed and cleaned reads were then mapped to the
mouse reference genome (mg38) using Bismark version 0.19.0 with the
following parameters: ” –bowtie2 –maxins 1000”.11 Differential
methylation analysis was performed using methylKit version 1.4.0 within
an R version 3.4.1 environment.12 Bismark alignments were processed via
methylKit in the CpG context with a minimum quality threshold of 10.
Coverage was normalized after filtering for loci with a coverage of at
least 5 reads and no more than the 99.9th percentile of coverage values.
The coverage was then normalized across samples and the methylation
counts were aggregated for 500nt windows spanning the entire genome. A
unified window set across samples was derived such that only windows
with coverage by at least one sample per group were retained.
Differential methylation analysis between conditional groups was
performed using the Chi-squared test and applying a qvalue (SLIM)
threshold of 0.05 and a methylation difference threshold of 25 percent.
Cell Culture: Human pulmonary artery smooth muscle cell
(PASMCs; Purchased from Lonza, USA) were maintained at 37°C under 5%
CO2 in smooth muscle basal media (Lonza, #CC-3181)
supplemented with growth factors (SMGM-2 smooth muscle singlequots kit,
Lonza, #CC-4149). Once cells reached approximately 70% confluence,
they were sub-cultured using 0.05% trypsin-EDTA (GIBCO, Cat #
25300-054, Thermo Fischer Scientific, Grand Island, NY) into 6-well
plates at about 3x105 cells/well.
Statistical analysis: Statistical analysis was performed using
GraphPad Prism 5 software. Values are presented as mean ± standard error
(SE). Statistical comparisons of samples were performed with Student’s t
test for comparing two groups. Multiple comparisons were performed by
one-way ANOVA followed by Sidak’s post hoc test. Difference with
P<0.05 between the groups was considered significant.