The Animal Experiment Committee of Mie University School of Medicine approved the study protocol (No. 20-34 and 20-35). All the animals were housed in climate-controlled conditions with 12 h light and 12 h dark cycle. Rats were fed rat chow containing 0.3% CLZ  or control chow without CLZ. Rat chow with and without CLZ was a gift from Otsuka Pharmaceutical Co., Ltd (Japan). 181 Seven-week-old male healthy male Sprague-Dawley rats weighing 185–245 g were purchased from Japan SLC, Inc. The rats were anesthetized with pentobarbital, placed on a ventilator, and euthanized by incising the abdominal aorta and exsanguination.
Seven-week-old male Sprague-Dawley rats (SLC, Japan) weighing 185-245 g were used. Rats were fed rat chow with or without CLZ one day before a single injection of MCT (60 mg/kg, Sigma) or saline and continued to be fed the same rat chow for another 21 days (Figure 1A). Each animal was randomly assigned to one of four groups: 1) a single injection of saline and rat chow without CLZ (Sal21/CLZ-) (n=10), 2) a single injection of saline and rat chow with CLZ (Sal21/CLZ+) (n=9), 3) a single injection of MCT and rat chow without CLZ (MCT21/CLZ-) (n=18), and 4) a single injection of MCT and rat chow with CLZ (MCT21/CLZ+) (n=14). MCT (60 mg/kg) [12,13,14,15,48,51,53] or the same volume of 0.9% NaCl was subcutaneously injected into the hind flank.
Seven-week-old male Sprague-Dawley rats (SLC, Japan) weighing 187-235 g were used. Rats were fed rat chow with or without CLZ beginning one day before the start of hypobaric CH exposure (air at 380 mmHg) and continued to be fed the same rat chow until the final day of ambient air or CH exposure (Figure 2A). Each animal was randomly assigned to one of four groups: 1) rats exposed to ambient air without CLZ (Air/CLZ-) (n=10), 2) rats exposed to ambient air with CLZ, (Air/CLZ+) (n=10), 3) rats exposed to CH without CLZ (CH/CLZ-) (n=14) and 4) rats exposed to CH with CLZ (CH/CLZ+) (n=14). Rats were exposed to hypoxia for 14 days and returned to ambient air after catheterization [6,8,9,10,11].
In the MCT28 model, each animal injected with saline or MCT (60 mg/kg) was randomly assigned to one of three groups (Figure 1B): Sal28/CLZ- (n=5), MCT28/CLZ- (n=8), and MCT28/CLZ+ (n=9). Rats were fed for 28 days after the injection of MCT as in the MCT21 model. In the MCT28 model, the rats were used to evaluate systolic right ventricular pressure (sRVP) under 45 mg/kg pentobarbital anesthesia and RVH and to obtain lung samples for protein and mRNA assays (Figure 1B). In the MCT21 and CH models, the rats were used to evaluate awake mean PAP (mPAP), RV/LV+S, and pulmonary vascular structural changes and to obtain lung samples for protein and mRNA assays (Figures 1A, 2A).
mPAP in MCT21 and CH, and sRVP in MCT28
At the end of 21 days after the MCT injection and 14 days of CH exposure, a pulmonary artery catheter (silastic tubing, 0.31 mm ID and 0.64 mm OD) was inserted via the right external jugular vein into the pulmonary artery by employing a closed-chest technique under 45 mg/kg pentobarbital anesthesia with no tail movement with stimulation [10,11,13] (Figures 1A, 2A). The left internal carotid artery was also cannulated. Twenty-four hours after the catheterization with the rat fully conscious, the mPAP and mean artery pressure (mAP) were recorded with a physiological transducer and an amplifier system (AP 620G, Nihon Kohden, Japan) once the rats were calm (Figures 1A, 2A).
In the MCT28 model, at the end of 28 days after MCT injection, sRVP was measured by the closed-chest technique under 45 mg/kg pentobarbital anesthesia, and then lung samples for protein and mRNA assays were obtained (Figure 1B).
Preparation of lung tissue for morphometric analysis and lung sampling for protein and mRNA assays
After the measurement of awake mPAP in the MCT21 and CH models, the rats were anesthetized with 50 mg/kg pentobarbital again and mechanically ventilated through tracheostomy. The abdomen was then incised, and the abdominal aorta was incised to cause blood loss and euthanasia. A midline sternotomy was performed to expose the heart and lung. The hilum of the right lung was ligated, and the right lung was excised and put into liquid nitrogen for real-time polymerase chain reaction (PCR) and Western blotting of whole lung tissue. Blood samples were collected for hematocrit measurement. A left lung section was prepared for morphometric analysis of the vasculature using the barium injection method [6,8,9,10,11,12,13,14] to identify peripheral pulmonary arteries. Briefly, the left pulmonary artery was injected with a hot radiopaque barium-gelatin mixture at 100 cm H2O pressure [6,8,9,10,11,12,13,14]. After injection, the lung was distended and perfused through the tracheal tube with 10% formalin at 36 cm H2O pressure for 72 hr. Sections were stained for elastin by the Van Gieson method. The right ventricle (RV) of the heart was dissected from the left ventricle plus septum (LV+S) and weighed separately. The heart weight ratio (RV/LV+S) was calculated to assess RVH. Lung sampling for MCT28 was described above.
Morphometric analysis of pulmonary arteries
Light microscope slides were analyzed without previous knowledge of the treatment groups. All barium-filled arteries in each tissue section were examined at x 400, for an average of 220 arteries per section (110-340 arteries per section). Each artery was identified as being one of two structural types for the presence of muscularity: muscularized (with a complete medial coat, incomplete medial coat, or only a crescent of muscle being present) and nonmuscular (no muscle apparent) [6,8,9,10,11,12,13,14]. The percentages of muscularized arteries (%Muscularization) in peripheral pulmonary arteries with an external diameter between 15 and 50 µm and those between 51 and 100 µm were calculated. For muscular arteries between 51 and 100 µm in diameter and those between 101 and 200 µm in diameter (an average of 18 arteries (5-20 arteries) were found per section), the wall thickness of the media (distance between external and internal elastic laminae) was measured along the shortest curvature, and the percent medial wall thickness (%MWT) was calculated [6,8,9,10,11,12,13,14].
Western blotting for eNOS, peNOS, AKT, IκB, and HMGB-1.
For Western blotting, lung samples were randomly selected from the MCT21, MCT28, and CH models, where all pooled lung samples could not be used because of the number of gel lanes. Samples were homogenized, and the supernatant was standardized to 3.0 mg/ml. Thirty micrograms of total protein from each sample was subjected to SDS-PAGE on 10% polyacrylamide gels (Nacalai, Japan) and blotted onto a PVDF membrane (Amersham Hybond-PR, GE Healthcare). Blots were blocked for 1 hr in 5% skimmed milk diluted in 0.1% TBST (Tris Buffered Saline Tween) followed by incubation overnight at 4°C in primary antibody diluted in Can Get Signal Immunoreaction Enhancer Solution 1 (Toyobo Co. Ltd., Japan). Six different primary antibodies were used, including endothelial nitric oxide synthase (eNOS) (BD Transduction Laboratories; G10296, lot 21527, 1:4000 dilution), phosphorylated eNOS (peNOS) (Cell Signaling; phospho-eNOS, ser-1177 #9751, 1:2000 dilution), serine-threonine protein kinase (AKT) (Cell Signaling; #4814S, 1:2000 dilution), phosphorylated AKT (pAKT) (Cell Signaling; #460P, 1:2000 dilution), IκB-α (Cell Signaling; #4814S, 1:2000 dilution), high-mobility group box-1 (HMGB1) (Cell Signaling; #3935S, 1:2000 dilution), and beta-actin (Sigma; A5441, 1:200,000 dilution). Next, the blots were incubated in secondary antibody (Amersham NA 931, 1:20,000 dilution) diluted in Can Get Signal Immunoreaction Enhancer Solution 2 (Toyobo Co. Ltd., Japan) for 1 hr at room temperature and in Immobilon Western Chemiluminescent HRP Substrate (Millipore Corporation, USA) for 5 min. Luminescent signals were captured digitally, and densitometry was performed using Multi Gauge Ver. 3.0 (Fujifilm, Science Laboratory 2005, Japan). Each target protein was normalized to β-actin, and the relative fold change compared to the control group (100%) was calculated.
cDNA preparation and PCR
eNOS, AKT, and monocyte chemotactic protein-1 (MCP-1) mRNA levels in whole lung tissue were determined by real-time PCR. After the extraction of total RNA from whole lung tissue using TRIzol reagent (Invitrogen, USA), cDNA synthesis was performed with ReverTra Ace (Toyobo Co., Ltd., Biochemical Operations Department, Osaka, Japan). cDNA samples (15 ng of total RNA) were amplified with a StepOne Plus Real Time PCR System (Applied Biosystems). The sequences of the primer pairs are listed in Table 1. Relative quantification was performed with the comparative ∆∆Ct method by normalization to β-actin mRNA.
Sixty rats (7-week-old male Sprague-Dawley rats (SLC, Japan)) were used. Each rat was randomly assigned to one of two groups: 30 rats (weighing 213-234 g) fed rat chow without CLZ and 30 rats (195-233 g) fed rat chow with CLZ (Figure 2B). The rat chow with CLZ (including 0.3% CLZ) was the same as that used with the MCT21, MCT28, and CH models. One day after the assignment of the feeding group, all rats were subcutaneously injected with MCT (60 mg/kg). Food and water were provided ad libitum. The number of rats alive was counted every day, and the Kaplan-Meier survival curve was obtained until 30 days after the injection of MCT.
Values are expressed as the means ± SE. When more than two means were compared, one-way analysis of variance was used. When significant variance was found, Fisher’s protected least significant difference test was employed to establish which groups were different. Survival was evaluated by the Breslow-Gehan-Wilcoxon test in StatView5.0R. Differences were considered significant at P<0.05.