Figure Legends:
Figure 1: Daily injection of a novel G6PD inhibitor, PDD4091,
decreased right ventricle pressure, hypertrophy, and pulmonary artery
remodeling in mice elicited by hypoxia and hypoxia+Sugen5416. A) A
schematic showing various treatment protocols in C57BL/6 mice. B)
Original tracing showing right ventricle pressure in mice treated with
and without PDD4091 (1.5
mg.kg-1.day-1) for 3 weeks in
normoxia and hypoxia, and mice treated with Sugen5416 (20
mg.kg-1) once a week. Right ventricle systolic (RVSP;
C) and diastolic (RVDP; D) were reduced by PDD4091 treatment for 3 weeks
to hypoxic mice. N=6-11 in each group (male=3-6 and female=1-5).
Statistical analysis was performed using Two-way ANOVA and Sidak’s test
for multiple comparisons.
Figure 2: Right ventricle pressure and hypertrophy is less in
G6PDS188F compared to wild-type rats exposed to
Sugen/Hypoxia/Normoxia. A) A schematic showing protocol of exposing rats
to Sugen5416 (20 mg.kg-1) followed by hypoxia (10% O2)
for 3 weeks and normoxia (ambient air) for 5 weeks. B, C) Right
ventricle systolic (RVSP) and diastolic (RVDP) pressure increased more
in wild-type rats as compared to G6PDS188F rats
exposed to Su/Hx/Nx than normoxic control. D) Right ventricle
hypertrophy indicated as RV-to-LV+S ratio (Fulton’s Index) increased in
wild-type compared to G6PDS188F rats exposed to
Su/Hx/Nx than normoxic control. N=4 in each group. All male rats were
used in the experimental groups because G6pd is a X-linked gene.
Statistical analysis was performed using Two-way ANOVA and Sidak’s test
for multiple comparisons.
Figure 3 : G6PD inhibitor, PDD4091, relaxed pre-contracted PA,
decreased PASMC growth, and rescinded occlusive lesion in PA. A)
Pulmonary arterial rings were contracted with KCl (30 mM) and
application of PDD4091 relaxed the pre-contracted arterial rings in
dose-dependent manner, (N=6 in each dose). B) Application of PDD4091 (1
µmol/L) to human pulmonary artery smooth muscle cells for 48 hours
decreased growth of cells cultured in 21% O2, N=6. C)
Hypoxia (3% O2; N=6) and Sugen (1 µmol/L; N=6) as
compared to normoxia-control (21% O2; N=6) increased
growth of control human pulmonary artery smooth muscle cells, and
application of PDD4091 (1 µmol/L) to cells in hypoxia (N=6) for 48 hr
reduced cell growth. D) Immunofluorescent micrograph shows occluded
pulmonary artery in lungs of mice exposed to hypoxia+Sugen5416 (SU), and
occluded pulmonary arteries were not present in lungs of
hypoxia+Sugen5416 (SU) mice treated with PDD4091 for 3 weeks. N=4 in
normoxia; hypoxia+Sugen5416 (SU) and hypoxia+Sugen5416 (SU)+4091 groups.
*P<0.05 vs 3x10-9 M in panel A.
*P<0.05 vs control or normoxia (Nx), and #P<0.05 vs
hypoxia in panel B and C. Statistical analysis was performed by One-way
ANOVA in panel A and C, and by Student’s t -test in panel B.
Figure 4 : Differential gene expression in lungs of mice exposed
to hypoxia and hypoxia+Sugen. A) Venn diagram of whole-genome RNA-seq
analysis demonstrate 3 genes are common in significantly up regulated
cohort and 1085 genes are common in significantly down regulated cohort
in lungs of mice exposed to hypoxia (Hx) and hypoxia+Sugen (Hx+SU)
compared to normoxia-control (Nx). B) Transcription factor binding site
enrichment analysis using oPPOSUM revealed TCFCP2L1 and KLF4 in Hx vs Nx
and HIF1A::ARNT and KLF4 in Hx+SU vs Nx are the most enriched TFBS in
genes up regulated category, and REST and HOXA5 in Hx vs Nx and HOXA5
and PDX1 in Hx+SU vs Nx are the enriched TFBS in genes down regulated
category in mice lungs. C) Heat map of RNA-seq results demonstrateSufu and Cyp1a1 genes are most up regulated in lungs of
mice exposed to Hx+SU vs Nx but not to Hx vs Nx, and Tubg2 andSv2b genes are most down regulated in lungs of mice exposed to
Hx+SU vs Nx and to Hx vs Nx. N=3 in each group. RNA-seq was performed on
three lungs in each group. Male=2 and female=1. The Benjamini-Hochberg
method was applied for multiple test correction (FDR<0.05).
Figure 5 . DNA methylation in lungs of mice exposed to hypoxia
and hypoxia+Sugen5416. A) Expression of genes that encode ten-eleven
translocation 1 demethylase (Tet1 ) and DNA methyltransferase 3b
(Dnmt3b ) is significantly decreased in lungs of mice exposed to
hypoxia (Hx) and hypoxia+Sugen5416 (Hx+SU). Methylation of the DNA in
lungs of mice exposed to normoxia, hypoxia and hypoxia+SU was determined
by Reduced Representation Bisulfite Sequencing method. B, C) Pie graph
of differentially methylated CpG regions demonstrate less regions are
hyper- than hypo-methylated in lungs of mice exposed hypoxia (Hx) and
hypoxia+Sugen (Hx+SU) vs normoxia-control (Nx). D) Cyp1a1 andKcng3 genes were hypomethylated in lungs of Hx and Hx+SU mice.
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.
Figure 6 . Expression of Cyp1a1 and Sufu genes is
decreased by G6PD inhibitor, PDD4091. A, B) Real-time PCR results
disclosed that Cyp1a1 and Sufu expression are increased in
lungs of mice exposed to Hx+SU but not to Hx, and PDD4091 treatment
decreased Cyp1a1 and Sufu . N=4, lungs from 3 different
mice (male=2 and female=1) were used for RNA-seq analysis in each group,
and N=5, lungs 5 different mice (male=3 and female=2) were used for qPCR
analysis in each group. C) Expression of CYP1A1 and SUFUincreased in human pulmonary artery smooth muscle cells cultured in
hypoxia (3 % O2), but not in normoxia (21%
O2), by Sugen5416 (SU: 1 µmol/L). Application of PDD4091
(1 µmol/L) to cells for 48 hr rescinded their elevated expression ofCYP1A1 and SUFU . N=6 in each experimental condition.
Statistical analysis was performed using Two-way ANOVA and Sidak’s test
for multiple comparisons.