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.