Nicotinamide Adenine Dinucleotide Phosphate Oxidase–Mediated Redox Signaling and Vascular Remodeling by 16α-Hydroxyestrone in Human Pulmonary Artery Cells

Supplemental Digital Content is available in the text.


Lucigenin-enhanced chemiluminescence
ROS generation was assessed in cell lysates by lucigenin-enhanced chemiluminescence assay as previously described 3, 6 . Control hPASMC, PAH-hPASMCs and hVSMCs were stimulated with E2 or 16αOHE1 for 5 minutes to 48 hours. In some experiments, cells were pre-exposed for 30 minutes to ML171, GKT137831, gp91ds-tat or scrambled peptide, tempol, MPP or PHTPP. Inhibitor studies were carried out at the peak time points for ROS production, 4hours stimulation for E2 and 30 minutes for 16αOHE1 treatment, respectively. Cells were washed with ice-cold PBS and harvested in lysis buffer (20mmol/L of KH2PO4, 1 mmol/L of EGTA, 1μg/mL of aprotinin, 1μg/mL of leupeptin, 1μg/mL of pepstatin, and 1mmol/L of PMSF). 50µl of sample was added to a suspension containing 175μl of assay buffer (50mmol/L of KH2PO4, 1mmol/L of EGTA, and 150mmol/L of sucrose) and lucigenin (5μmol/L). Luminescence was measured with a luminometer (AutoLumat LB 953, Berthold) before and after stimulation with NADPH (100µmol/l). A buffer blank was subtracted from each reading. Superoxide anion production was inhibited by tempol and was expressed as relative luminescence units (RLU)/µg protein, relative to vehicle control conditions.

Amplex Red assay
Hydrogen peroxide (H2O2) was assessed in cell lysates using the fluorescence assay Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit (Life Technologies, Carlsbad, CA, USA) in accordance with manufacturer's instructions and as we have previously described 2 . Briefly, Amplex Red (50 μmol/L) and horseradish peroxidase (0.1 U/mL) were added to the cellular samples. Fluorescence readings were made in a 96-well plate at Ex/Em = 530/590 nm using 50 µl samples harvested in protein lysis buffer. H2O2 production was normalized to protein concentration. The results are expressed in arbitrary units per micro-gram protein, relative to vehicle control conditions.

Immunoblotting
Proteins were extracted from cells and mouse pulmonary artery and immunoblotting was performed as we previously described 7 . All antibodies were used at 1:1000 dilution unless otherwise stated. After incubation with HRP-conjugated secondary antibodies, signals were revealed by chemiluminescence (WestPico, Pierce), visualized by autoradiography, and quantified densitometrically with open-source software ImageJ. Anti--actin antibody (1:10,000) was used as protein loading control.
Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) activity assay To assess nuclear accumulation of Nrf2, samples were prepared according to the manufacturer's protocol using a nuclear extract kit (Active Motif, Carlsbad, CA). Nuclear preparations (10μg) were used for the TransAM Nrf2 ELISA kit (Active Motif, Carlsbad, CA) to measure DNA binding of activated Nrf2 nuclear protein, as determined by absorbance at 450nm, following manufacturer's instructions and as previously described.

DNA synthesis by BrdU incorporation
Cell proliferation by E2 and 16αOHE1 was measured by BrdU incorporation using a Proliferation Assay kit (Calbiochem, Darmstadt, Germany), according to the manufacturer's instructions. Cells were seeded onto a 96-well plate and starved overnight before stimulation with E2 or 16αOHE1 in the absence or presence of ML171, GKT137831 or gp91ds-tat. Cells were incubated with BrdU for 24 hours. Absorbance was obtained at dual wavelength (450nm and 595nm) with a spectrophotometer (Spectra Max; Molecular Devices, Sunnyvale, CA, USA). The results were normalized as percent of control vehicle conditions.

Hypoxia-induced pulmonary hypertension in Nox1-/-and Nox4-/-mice
In studies involving the influence of the estrous cycle on non-reproductive functions, vaginal smear cytology is used to determine the cycle phases 8 . This was carried out prior to normoxic or hypoxic exposure to ensure this experimental model was commenced on the same cycle day for all mice.    GAPDH  GAGTCAACGGATTTGGTCGT  TTGATTTTGGAGGGATCTCG  Nox1  TCACCAATTCCCAGGATTGA  TGTGGTCTGCACACTGGAAT  Nox4  TGCAGCAAGATACCGAGATG  GTGATCATGAGGAATAGCAC  p47phox  AGTCCTGACGAGACGGAAGA  TACATGGACGGGAAGTAGCC  p67phox  AAGCTGTTTGCCTGTGAGGT  CTGCTTCCAGACACACTCCA  p40phox  TCCTCCTCAGTCGGATCAAC  TGATGGTGCTGATGGTGTCT  NoxA1 CATGATGCCAGGTCCCTAAT

Supplemental
GCAGAAACTTCAACCCGATAA GAGCGGGGCGGAGAGT Primers targeted to the above genes were designed using Primer 3 software online, and were used to assess gene expression. *** ‡ ‡ ‡ S1: CYP1B1 inhibition by TMS has no effect on 16αOHE1-induced ROS formation. Cells were exposed to TMS (100nmol/L) prior to 30 minutes stimulation with 16αOHE1 (A      Transcript levels of CYP1B1 in female WT and Nox1-/-mouse pulmonary artery tissue (B) and in WT and Nox4-/-mouse pulmonary artery tissue (C). Results are mean ±SEM of 4 experiments, in triplicate. Graphs represent the protein expression relative to β-actin or mRNA expression relative to GAPDH. *p<0.05, vs. normoxic WT; ‡p<0.05, vs normoxic Nox4-/-; †p<0.05, vs. hypoxic WT, determined by ANOVA with Tukey's post-hoc test.