All of the protocols and surgical procedures were approved by the Institutional Animal Use Committee of the Third Military Medical University and were in accordance with the National Institutes of Health and the American Physiological Society guidelines. Adult male Wistar rats (6 – 7 weeks old, 220 – 250 g) were placed for 21 days in a chamber that was depressurised to 380 mmHg with a 12-hour light–dark cycle. Age-matched controls were maintained in normal room air. Each group consisted of 15 experimental animals. The methods that were used to isolate the rat lungs were similar to those previously reported . In brief, prior to lung isolation, mean pulmonary arterial pressures were measured as previously described . After euthanizing the rats, the thorax was immediately opened and the heart and lungs were removed. The hearts were dissected to remove the right ventricle (RV) free wall and the left ventricle plus septum (LV + S), and the weight ratio of RV/(LV + S) was used as an index of RV hypertrophy. The distal intrapulmonary arteries were dissected from the lungs and frozen in liquid nitrogen for subsequent examination.
Morphological preparation and examination
The lungs was perfused with 4% paraformaldehyde (PFA), inflated by infusion of 4% PFA at a constant pressure of 25 cm H2O through the cannula inserted in the trachea, fixed in 4% PFA overnight at 4°C and then embedded in OCT, and subsequently cut into 10-μm-thick sections for hematoxylin and eosin staining. Following H&E staining, Images of individual pulmonary arteries were captured using a digital camera, mounted on a light microscope, and linked to a computer. the ratio of vessel wall area to total area (WA%) and the ratio of pulmonary arteriole wall thickness to vascular external diameter (WT%) were measured using the Image-Pro Plus 5.1 software.
Quantitative real-time polymerase chain reaction (qRTPCR)
Total RNA from the intrapulmonary arteries (isolated as mentioned above) was isolated using the RNA simple Total RNA Kit (Tiangen), according to the manufacturer’s protocol. Then, the total RNA was reverse-transcribed to cDNA using the PrimeScript® RT reagent kit (TAKARA). Real-time PCR was performed using the SYBR® Premix Ex Taq™ II kit (TAKARA). The primer sequences for STIM1 were 5′-CGTCCGCAACATCCACAAG-3′ (forward) and 5′-CCATAGGTCCTCCACGCT-3′ (reverse). The primer sequences for β-actin were 5′-ACGGTCAGGTCATCACTATC-3′ (forward) and 5′-TGCCACAGGATTCCATACC-3′ (reverse). The amplification conditions consisted of 1 cycle at 95°C for 30 s and 40 cycles of 95°C for 5 s, 60°C for 20 s, and 72°C for 15 s. Melting curve analyses were performed at conditions of 95°C for 1 min and then 55°C for 1 min, which were followed by 80 increments of +0.2°C at 10-s intervals. The relative concentrations of each transcript were calculated using the standard curve method.
The isolated lungs were formaldehyde fixed (4% in PBS), cryoprotected with 30% sucrose in PBS, embedded in OCT media, and then frozen. Cryostat sections (10 μm) were permeabilised and blocked for nonspecific binding and then incubated in 0.2% gelatin in PBS with the following primary antibodies at 4°C overnight: mouse monoclonal anti-STIM1 (1:100) (BD Bioscience) and rabbit polyclonal anti-α-smooth muscle actin (1:250) (ABcam). The secondary antibodies [anti-mouse Cy3 and anti-rabbit FITC (1:500) (Biyuntian, China)] were prepared in 0.2% gelatin in PBS and were applied to the sections for 1 h at 37°C. Nuclei were stained using DAPI (1:1,000 in PBS; Biyuntian, China). Images were taken using a confocal laser scanning microscope (Leica TCS SP5, Germany).
Isolation and culture of PASMCs
For isolation of the PASMCs, the adventitia of freshly excised distal (>4th generation) intrapulmonary arteries from adult male Wistar rats were removed. Vascular segments were then cut open, and the endothelium was removed by gently scraping the luminal surface of the vessel. Rat PASMCs were cultured as previously described . In brief, the arteries were allowed to recover for 30 min in cold (4°C) physiological salt solution (PSS) that contained 130 mM NaCl, 5 mM KCl, 1.2 mM MgCl2, 10 mM HEPES, and 10 mM glucose. This was followed by 20 min in reduced-Ca2+ PSS (20 μM CaCl2) at room temperature. The tissue was then digested at 37°C for 20 min in reduced-Ca2+ PSS containing collagenase (type I, 1,750 U/ml), papain (9.5 U/ml), bovine serum albumin (2 mg/ml), and dithiothreitol (1 mM). Cells were grown in DMEM supplemented with 10% FBS and were passaged at a 1:3 ratio with trypsin treatment. The purity of the PASMCs in the primary cultures was confirmed using the specific mAb against smooth muscle α-actin. Cells that had been passaged 3 to 8 times and were at 80% confluence were used for all experiments.
Small interfering RNA (siRNA) targeting STIM1 (siSTIM1) were designed as previously described  and synthesised by Genepharma. Scrambled (nonsense) siRNA (Genepharma) was used as negative control siRNA. Cells were transfected with siSTIM1 or Scrambled siRNA (final concentration of siRNA was 90 nM) (Invitrogen, Carlsbad, CA) using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. After 24 h, Cell growth was arrested by replacing medium with serum-free DMEM for 24 h under normoxic conditions. Growth-arrested cells were further incubated under either normoxic(21% O2) or hypoxic conditions (3% O2) for 24 h.
Forty-eight hours after transfection, cells were collected for protein isolation. The cultured cells were washed twice with ice-cold PBS and lysed on ice in RIPA lysis buffer containing freshly added protease and phosphatase inhibitor cocktails. After 15 min of incubation, the cell lysate was collected by centrifuging the cells for 5 min(16,000 × g) at 4°C. For nuclear and cytoplasmic fractions, adherent cells were washed in PBS, and the cytoplasmfraction was prepared by the addition of buffer C (10 mM Tris, pH 7.6, 10 mM KCl, 1.5 mM MgCl2, 1% (v/v) Triton X-100, 1 mM dithiothreitol, 0.2 mM Na3VO4, 0.4 mM phenyl-methylsulfonyl fluoride, 10 μg/ml leupeptin, and 0.2 mM NaF). After a 15-min incubation on ice, lysates were spun for 5 min (10,000 × g) at 4°C. Supernatant containing the cytoplasm fraction was saved, and the pellet, containing the nuclear fraction, was washed once with buffer C and resuspended in buffer N (20 mM Tris, pH 7.6, 160 mM KCl, 1.5 mM MgCl2, 10% (v/v) glycerol, 1 mM dithiothreitol, 0.2 mM Na3VO4, 0.4 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, and 0.2 mM NaF). Nuclear lysates were incubated for 30 min on a rotating platformat 4°C and spun (16,000 × g) for 15 min at 4°C. The amount of total protein was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). An equal amount of total protein (30-50 μg) was loaded and separated by SDS-PAGE. The protein was transferred to polyvinylidene difluoride membranes and was then blocked and probed with the appropriate antibodies. Monoclonal Abs against STIM1 (BD Bioscience), Lamin B1 (Santa Cruz) and β-actin (Santa Cruz) or a polyclonal antibody against NFATc3 (Santa Cruz) were used as primary Abs. The membranes were then washed for 15 min 3 times and incubated with horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG for 1 h. Bound antibodies were detected using an enhanced chemiluminescence system following the manufacturer’s instructions. Densitometric signals were quantified by Quantity One software.
Determination of cell proliferation
PASMC proliferation was quantified by [3H]-thymidine incorporation, as described previously . Briefly, 1 μCi/well [3H]-thymidine was added for the final 6 hours of cell culture, after which the cells were removed from the wells with trypsin digestion. The incorporated [3H]-thymidine was precipitated with 10% trichloroacetic acid and counted using a liquid scintillation counter. The experiments were repeated three times and results from five wells per experiment were determined and expressed as the average of the counts.
Cell cycle and DNA analyses
PASMCs were harvested by trypsin-EDTA treatment and were fixed in 70% ethanol. The ethanol was removed, and the cells were incubated in PBS containing RNase at 37°C for 30 min. Next, the cells were stained with propidium iodide (50 μg/ml) and suspended in PBS for 30 min on ice. DNA fluorescence was measured by flow cytometry using an EPICS XL cytometer (Beckman Coulter, CA).
Measurement of SOCE
The [Ca2+]i in PASMCs was measured using the Ca2+-sensitive fluorescent indicator fluo-4/AM. Cells were loaded with fluo-4 at 37°C for 30 min using a Ca2+-free physiological salt solution (D-HANKS) containing 5 μM fluo-4/AM. The fluo-4-loaded cells were then perfused with a Ca2+-free physiological salt solution containing the following components: 0.5 mM EGTA (Sigma Chemical, St Louis, MO)to chelate residual Ca2+, 5 μM nifedipine (Sigma Chemical, St Louis, MO) to prevent calcium entry through L-type voltage-operated Ca2+ channels (VOCC), and 10 μM cyclopiazonic acid (CPA; Sigma Chemical) to deplete SR Ca2+ stores. The [Ca2+]i was determined before and after the restoration of extracellular [Ca2+] to 2.5 mM. SOCE was evaluated by measuring the peak increase in [Ca2+]i caused by the restoration of extracellular Ca2+ in the continued presence of nifedipine and CPA.
Cells were fixed for 30 min at room temperature with 4% formaldehyde in Dulbecco's phosphate-buffered saline (DPBS). Next, the cells were incubated with 0.2% Triton X-100 in DPBS for 15 min at room temperature. The cells were blocked with blocking solution (10% goat serum in DPBS) for 1 h and then incubated with the primary antibodies (NFATc3, sc-8321, Santa Cruz Biotechnology, Santa Cruz, CA, USA) for 1 h at room temperature followed by the fluorescent-conjugated secondary antibody (FITC-conjugated AffiniPure goat anti-rabbit IgG, Beijing Zhongshan Golden Bridge Biological Technology Company, Beijing, China) for 90 min at room temperature. Nuclear staining was performed using DAPI (Biyuntian, China). The fluorescence was examined using a Leica laser scanning confocal microscope (Leica TCS SP5, Germany).
Numerical data were expressed as the means ± SEM. The SPSS10.0 software was used for the statistical analysis. An ANOVA was used with Scheffe’s multiple comparison tests for multiple groups and student’s t-test was used for two groups. P values < 0.05 were regarded as statistically significant.