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Figure 3 : G6PD inhibitor, PDD4091, relaxed pre-contracted PA, decreased PASMC growth, and rescinded occlusive lesion in PA.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.