The selective PGI2 receptor agonist selexipag ameliorates Sugen 5416/hypoxia-induced pulmonary arterial hypertension in rats.

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by a progressive increase in pulmonary artery pressure due to an increase in vessel tone and occlusion of vessels. The endogenous vasodilator prostacyclin and its analogs are used as therapeutic agents for PAH. However, their pharmacological effects on occlusive vascular remodeling have not been elucidated yet. Selexipag is a recently approved, orally available and selective prostacyclin receptor agonist with a non-prostanoid structure. In this study, we investigated the pharmacological effects of selexipag on the pathology of chronic severe PAH in Sprague-Dawley and Fischer rat models in which PAH was induced by a combination of injection with the vascular endothelial growth factor receptor antagonist Sugen 5416 and exposure to hypoxia (SuHx). Oral administration of selexipag for three weeks significantly improved right ventricular systolic pressure and right ventricular (RV) hypertrophy in Sprague-Dawley SuHx rats. Selexipag attenuated the proportion of lung vessels with occlusive lesions and the medial wall thickness of lung arteries, corresponding to decreased numbers of Ki-67-positive cells and a reduced expression of collagen type 1 in remodeled vessels. Administration of selexipag to Fischer rats with SuHx-induced PAH reduced RV hypertrophy and mortality caused by RV failure. These effects were probably based on the potent prostacyclin receptor agonistic effect of selexipag on pulmonary vessels. Selexipag has been approved and is used in the clinical treatment of PAH worldwide. It is thought that these beneficial effects of prostacyclin receptor agonists on multiple aspects of PAH pathology contribute to the clinical outcomes in patients with PAH.

Introduction

Pulmonary arterial hypertension (PAH) is a lethal disease characterized by a progressive increase in pulmonary artery pressure, which causes right ventricular (RV) hypertrophy leading to right heart failure [1]. The increase in pulmonary artery pressure is caused by an imbalance in the regulation of vessel tone and occlusion of the vessels by excessive proliferation of pulmonary arterial smooth muscle cells in the lumen [2].

Prostacyclin is one of the most important endogenous pulmonary vasodilators. The prostacyclin receptor plays an important role in the relaxation of vessel tone and in the regulation of the proliferation of pulmonary arterial smooth muscle cells. Therefore, prostacyclin and its analogs are widely used as therapeutic agents for the treatment of PAH [3-5]. Selexipag is a recently developed orally available and selective prostacyclin receptor agonist with a non-prostanoid structure [6]. In the phase III trial GRIPHON, the selexipag treatment group showed a significant reduction in the risk of the primary composite endpoint of death or a complication related to PAH compared with placebo [7]. Selexipag has been approved for the treatment of adult patients with PAH in the US, the EU and Japan [8].

In spite of the great success of prostacyclin receptor agonists as therapeutic agents for PAH, their pharmacological effects on the pathology of PAH, such as complex occlusive vascular remodeling and mortality risk associated with RV failure, have not yet been elucidated. The Sprague-Dawley (SD) rat model in which PAH is induced by a combination of subcutaneous injection of a vascular endothelial growth factor inhibitor, Sugen 5416, and exposure to hypoxia (SuHx) can reproduce PAH-specific complex occlusive vascular remodeling such as plexiform lesions [9,10]. This pathological feature is not observed in other commonly used PAH animal models which are induced by administration of monocrotaline or by only exposure to hypoxia [11]. The SD SuHx model also develops severe RV hypertrophy, systolic and diastolic RV dysfunction, and increased glucose uptake in the RV [12]. These progressive RV failure phenotypes closely resemble those of patients with PAH. In consideration of these facts, the SD SuHx model is regarded as one of the best rodent models for studying PAH. Recently some groups reported that the phenotypes induced by SuHx vary among rat strains. For example, in Wistar Kyoto rats SuHx induces not only occlusive vascular remodeling but also emphysema, whereas in SD rats SuHx induces only occlusive vascular remodeling [13,14]. In Fischer rats, SuHx induces severe RV failure similar to those induced in SD rats, but whereas SD SuHx rats show good survival SuHx Fischer rats tend to die of the failure of RV adaptation to increasing afterload. For these reasons, we chose the SD SuHx rat model to assess the effect of selexipag on hemodynamics and occlusive vascular remodeling and the Fischer SuHx rat model to assess its effect on mortality by RV failure.

The efficacy of the prostacyclin receptor agonists iloprost and treprostinil have been assessed in the SD SuHx model. They improve the hemodynamics but do not show ameliorative effects on pulmonary vascular remodeling [15,16]. We have previously reported that MRE-269, an active metabolite of selexipag, has potent vasodilator effects on lung vessels compared with other prostacyclin receptor agonists due to its high selectivity [17]. We hypothesize that selexipag can cause potent vasodilation even in the severe PAH of the SuHx model and improve the pathological features of PAH, so in the present study we investigated the pharmacological effects of selexipag on the pathological features of PAH in the SD SuHx rat model and mortality by RV failure in the Fischer SuHx rat model.

Materials and Methods

Animals

All animal procedures were approved by the Committee for the Institutional Care and Use of Animals of Nippon Shinyaku Co., Ltd., which are based on the Law for the Humane Treatment and Management of Animals (Law No. 105, 1 October 1973, as revised on 1 June 2006). Six-week-old male SD rats (Japan SLC, Shizuoka, Japan) were used for all experiments except the survival experiment, in which six-week-old male F344/DuCrlCrlj (Fischer) rats (Japan Charles River, Yokohama, Japan) were used. The animals were housed three per cage in a room maintained at 20–26°C and 35–75% relative humidity with an alternating 12-h light/dark cycle (the lights came on automatically at 8:00 a.m.) and allowed free access to pellet chow (F-2; Funabashi Farm, Chiba, Japan) and tap water. Animals were allocated to groups by their body weights the day before the start of treatment in each experiment. The animals were well cared for by the animal care personnel and by the veterinarian at our institution. The condition of the animals was checked twice a day. Humane endpoints were used during all experiments. If any animals had shown either (1) persistent crouching or noisy breathing or (2) a loss of body weight greater than 20%, they would have been euthanized by the use of carbon dioxide to avoid suffering. However, no animals met either of those criteria during any of the experiments.

U46619-induced elevation of right ventricular systolic pressure (RVSP)

Rats were anesthetized by subcutaneous injection of ethyl carbamate (Wako, Osaka, Japan) at 1.2 g/kg. The rat was laid on its back and its temperature was maintained at 37°C with an animal blanket controller (model ATB-1100; Nihon Kohden, Tokyo, Japan) throughout the measurement. A polyethylene cannula (PE-50; Becton, Dickinson and Company, Franklin Lakes, NJ) filled with heparinized saline was inserted into the right femoral artery for measurement of the mean arterial pressure (MAP) and heart rate (HR) and another was inserted into the right femoral vein for intravenous administration of U46619 (Cayman Chemical, Ann Arbor, MI), which was dissolved in dimethyl sulfoxide (5 mg/mL) and diluted with saline (60 μg/mL). Another cannula was inserted into the right jugular vein for measuring the RVSP. For administration of selexipag, a silicon tube was inserted into the duodenum. Selexipag (synthesized by Nippon Shinyaku Co., Ltd.) was suspended in 0.5% (w/v) methylcellulose (Metolose SM-400; Shin-Etsu Chemical, Tokyo, Japan) in distilled water (Otsuka Pharmaceutical, Tokushima, Japan). Acute elevation of RVSP was produced by continuous infusion of U46619. The infusion rate was adjusted within the range of 0.6–1.2 μg/kg/min to give an RVSP of 45–55 mmHg. After the RVSP had stabilized, the value of RVSP at 0 min was recorded and selexipag (3 mg/kg) or vehicle (0.5% methylcellulose solution) was administered intraduodenally (N = 8 per group). RVSP was measured and recorded for 120 min after administration of selexipag. At 10, 20, 30, 60, 90 and 120 min after administration, the 1-min average values of RVSP were calculated with the PowerLab software (ADInstruments, Bella Vista, Australia) and used as the measured values. The change in RVSP from its value at 0 min was calculated for the measurements from 10 to 120 min.

SuHx-induced PAH model

Sugen 5416 was synthesized by Nippon Shinyaku Co., Ltd., and suspended at 5 mg/mL in a buffer containing 0.5% (w/v) carboxymethyl cellulose sodium salt (Sigma-Aldrich, St. Louis, MO), 0.9% (w/v) sodium chloride (Nacalai Tesque, Kyoto, Japan), 0.4% (v/v) polysorbate 80 (Sigma-Aldrich) and 0.9% (v/v) benzyl alcohol (Nacalai Tesque). Six-week-old SD rats were subcutaneously injected with Sugen 5416 (20 mg/kg) and exposed to 10% oxygen for three weeks (SuHx) [18]. Thereafter they were returned to normoxia and maintained on normoxia. Normoxia rats were kept in room air during the experimental period. Vehicle-treated groups were orally administered 0.5% methylcellulose solution twice daily. Selexipag-treated groups were orally administered compound suspended in 0.5% methylcellulose solution as two daily injections. The treatment protocol for each study group is shown in Fig 2. In the early-stage study, SuHx rats were administered either selexipag (10 or 30 mg/kg) or vehicle for three weeks (from 3 weeks after Sugen 5416 injection to the end of the 6th week), then sacrificed to measure the outcomes (Fig 2A; N = 8 for the normoxia group and N = 10 for the other groups). In the late-stage study, SuHx rats were administered either selexipag (30 mg/kg) or vehicle for three weeks (from 5 weeks after Sugen 5416 injection to the end of the 8th week), then sacrificed to measure the outcomes (Fig 2B; N = 10 per group). The SuHx rats which were sacrificed at the beginning of the treatment were used as pre-treatment control groups. RVSP and RV hypertrophy were measured as outcomes 24 h after the final administration of selexipag. The left lungs of the rats were collected at the time of measurement of the outcomes and used for histological analysis.

Fig 2

Study design using the SuHx rat model.

Scheme of the early-stage study (A) and the late-stage study (B). Black arrowheads indicate the date for the assessment of the outcome parameters.

Measurement of arterial pressure and heart rate

The day before RVSP evaluation, the mean arterial pressure (MAP) and heart rate (HR) were measured with a tail-cuff indirect blood pressure meter (BP-98A; Softron, Tokyo, Japan). The conscious rat was placed in a holder maintained at 37°C and the cuff attached to its tail, and then the MAP and HR were measured.

RVSP measurements

Rats were anesthetized with isoflurane (Pfizer Inc., New York, NY) and mechanically ventilated (Dwyer SAR-830/P ventilator; CWE, Ardmore, PA). The rat was laid on its back and its body temperature was maintained at 37°C with an animal blanket controller throughout the experimental period. A polyethylene cannula filled with heparinized saline was inserted into the right jugular vein and advanced into the right ventricle for monitoring the RVSP. The cannula was connected to a non-isolated bridge amplifier (FE228 Octal Bridge Amp; ADInstruments), and the RVSP was recorded with a PowerLab 16/35 data acquisition system (ADInstruments). The system was carefully calibrated with a mercury manometer (201-50-22; Acoma, Tokyo, Japan) before each experiment.

Measurement of RV hypertrophy and tissue preparation

After RVSP evaluation, rats were euthanized by exsanguination from the abdominal aorta under 3% isoflurane anesthesia. The left lungs were removed, perfused with heparinized saline and then fixed in 10% neutral buffered formalin. The right ventricle (RV) and left ventricle plus septum (LV+S) were weighed separately and the ratio of RV to LV+S [RV/(LV+S)] was calculated.

Histological analysis

The embedding of the left lungs in paraffin and tissue staining were performed by Applied Medical Research Laboratory (Osaka, Japan). The stained sections were digitized with an Aperio CS2 Digital Pathology Scanner (Leica Biosystems, Nussloch, Germany) and analyzed with the Aperio ImageScope software (Leica Biosystems). To evaluate the occlusive vessel lesions, including medial wall hypertrophy, reaction/proliferation of the vascular endothelium, and concentric laminar neointimal lesions, small vessels whose outer diameters ranged from 30 to 50 μm (30 vessels per section) were assessed. Specifically, the occlusive ratio of the lumen was determined from the vessel outer diameter and the luminal diameter. An occlusive vessel lesion was defined as a partial (>50%) or full obstruction of the lumen of a vessel [10,19]. For morphometric analysis, the medial wall thickness was assessed in arteries whose outer diameters ranged from 50 to 200 μm. The percent medial thickness of arteries was calculated with the formula [(outer diameter—inner diameter)/outer diameter] × 100 [10]. For immunohistochemical analysis, paraffin-embedded lung tissue slides were deparaffinized and hydrated. Sections were boiled in ethylenediaminetetraacetic acid buffer (pH 9.0) for 40 min to retrieve the Ki-67 epitope or incubated with proteinase K for 5 min at room temperature to retrieve the von Willebrand factor (vWF) or collagen type I epitope. After quenching endogenous peroxidase activity with 0.3% hydrogen peroxide in methanol, the sections were incubated with anti-Ki-67 antibody (diluted 1:1; Nichirei Biosciences, Tokyo, Japan) for 60 min at room temperature, anti-vWF (diluted 1:5000; Nichirei Biosciences) overnight at 4°C or anti-collagen type I antibody (diluted 1:100; Southern Biotechnology Associates, Birmingham, AL) overnight at 4°C. Immunoreactivity was visualized using 3,3'-diaminobenzidine (DAB). Apoptotic cells in the lung vessels were detected by terminal deoxynucleotidyl transferase mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL) assay using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (Merck, Darmstadt, Germany). The small vessels (outer diameter, 30–50 μm; 30 vessels per section) were assessed for the proportion of Ki-67-positive vessels and the expression of collagen type I. Vessels containing more than one cell stained with anti-Ki-67 antibody were counted as Ki-67-positive vessels. To evaluate the expression level of collagen type I, the vessel area was determined as the area encircled by the tunica adventitia and DAB-positive pixels in the vessel area were counted with Positive Pixel Count v9, a macro in the ImageScope software. Then the ratio of the number of positive pixels to the vessel area was calculated for each vessel and the average number of positive pixels per square micrometer for 30 vessels in each lung was used as the value for the individual section for statistical analysis [10,20,21].

Mortality and RV hypertrophy in SuHx-induced PAH Fischer rats

Six-week-old Fischer rats were subcutaneously injected with Sugen 5416 (20 mg/kg) and exposed to 10% oxygen for three weeks, after which they were returned to normoxia. The rats were treated with vehicle (0.5% methylcellulose solution) or selexipag (30 mg/kg) orally twice daily from day 22 to day 42 after Sugen 5416 subcutaneous injection (N = 10 per group). The general condition of the rats was checked twice daily. Observation was terminated on day 42 and all surviving animals were euthanized by exsanguination from the abdominal aorta under 3% isoflurane anesthesia to measure their RV hypertrophy.

Statistical analysis

All data were expressed as the mean ± S.E.M. and figures were drawn with GraphPad Prism 6 (GraphPad Software, San Diego, CA). In the evaluation of the U46619-induced elevation of RVSP, the selexipag-treated group was compared with the vehicle-treated group by repeated measures analysis of variance (ANOVA). When a significant difference was noted, the significant differences at each time point were analyzed by Student’s t-test between the two groups. In the SuHx models, statistical analysis was performed by Tukey’s test. Kaplan-Meier curves for survival rates were drawn with GraphPad Prism 6 and compared using log-rank analysis. RV hypertrophy in the SuHx Fischer model was analyzed by Student’s t-test. All statistical analyses were performed with SAS System Version 9.3 (SAS Institute Inc., Cary, NC) and EXSUS Version 8.1.0 (CAC Croit Corporation, Tokyo, Japan). A P value of less than 0.05 was considered statistically significant.

Results

Effect of selexipag on U46619-induced elevation of RVSP

Prior to investigating the effects of selexipag on the pathology of PAH, we verified the vasodilating activity of selexipag in lung and systemic vessels in an acute rat model of PAH induced by the thromboxane A2 agonist U46619. Thromboxane A2 is produced by activated platelets and induces platelet aggregation and vasoconstriction [22]. Its plasma levels are increased in patients with PAH, and activation of the thromboxane receptor on vascular smooth muscle cells leads to vasocontraction through the accumulation of inositol phosphates [23,24]. Accordingly, intravenous infusion of a thromboxane agonist, U46619, to rats quickly induces elevation of RVSP and this is used to create an acute PAH model [25,26]. Intravenous infusion of U46619 produced an elevation in RVSP from 35.5 ± 0.9 to 48.1 ± 0.7 mmHg. We have previously reported that the administration of 3 mg/kg selexipag causes a slightly increased heart rate but has no effect on systemic blood pressure in rats not infused with U46619 [27]; therefore we chose a dose of 3 mg/kg to investigate the effect of selexipag on RVSP. This elevation of RVSP was significantly reduced within 30 min by intraduodenal administration of selexipag at 3 mg/kg, and the effect persisted until the end of measurement at 120 min (Fig 1A). No significant effect of selexipag on the mean arterial pressure (MAP) or heart rate (HR) was observed at any time during the experiments (Fig 1B and 1C). The maximum reduction in RVSP was approximately 16% (7.9 mmHg).

Fig 1

Hemodynamic effects of selexipag in U46619-induced acute PAH model rats.

Effects of selexipag on right ventricular systolic pressure (RVSP) (A), mean arterial blood pressure (MAP) (B) and heart rate (HR) (C). The percentage change in RVSP, MAP and HR from the value at 0 min were calculated for each measurement until 120 min. Statistical analyses were performed using repeated measures ANOVA followed by Student’s t-test. **P<0.01 vs. vehicle. Values are means ± S.E.M. N = 8 per group.

Effects of selexipag on hemodynamic parameters, RV hypertrophy and obstructive pulmonary vascular remodeling in the early phase of SuHx rats

To investigate the pharmacological effects of selexipag on the pathology of PAH, we tested selexipag on both the early-pathology and the late-pathology phase in SuHx rats (Fig 2).

Subcutaneous injection of SD rats with Sugen 5416 followed by exposure to 10% oxygen for three weeks induced a significant increase in RVSP (from 37.6 ± 0.8 to 95.7 ± 4.2 mmHg; Fig 3A) and RV/(LV+S) (from 0.23 ± 0.01 to 0.63 ± 0.02; Fig 3B). The severity of PAH differs between the acute U46619-induced and the chronic SuHx-induced rat models. The increase in RVSP in the SuHx model (from 37.6 ± 0.8 to 141.1 ± 6.2 mmHg for SuHx 6w; Fig 3A) was much greater than in the acute model (from 35.5 ± 0.9 to 48.1 ± 0.7 mmHg). Therefore, the fact that the higher dose of 10 or 30 mg/kg/day selexipag was needed in our SuHx model compared with the single administration of 3 mg/kg in the acute model may have been due to the more serious development of RVSP in the SuHx model. For the experiment on the early-pathology phase, selexipag (10 or 30 mg/kg/day) or vehicle was administered orally for three weeks. At the end of the treatment, the RVSP of the vehicle-treated group (SuHx 6w) had further significantly increased (to 141.1 ± 6.2 mmHg), while the ratio RV/(LV+S) showed no further increase. In contrast, in the group treated with selexipag at 30 mg/kg, although RVSP still remained high, it significantly decreased to 102.1 ± 5.7 mmHg with no effect on MAP or HR (Fig 3A, 3C and 3D). In concert with the decrease in RVSP, RV/(LV+S) was significantly reduced by both doses of selexipag relative to that of the vehicle-treated group (to 0.56 ± 0.02 at 10 mg/kg and 0.50 ± 0.02 at 30 mg/kg; Fig 3B). Occlusive lesion in pulmonary vessels is observed in patients with PAH and in SuHx rats, and it causes an increase in pulmonary vascular resistance and an elevation of RVSP [1,18]. To investigate the effect of selexipag on occlusive lesion, we histologically evaluated the proportion of small vessels with occlusive lesions in the lungs of SuHx rats. The rate of occlusive lesions was 14 ± 2% of the vessels at the end of hypoxia (SuHx 3w), and it progressed to 55 ± 5% at six weeks after Sugen 5416 injection in the vehicle-treated group (SuHx 6w) (Fig 4A and 4B). The progression of occlusive lesions corresponds to that of the elevation of RVSP. The rate of occlusive lesions was significantly reduced to 26 ± 7% in the group treated with 30 mg/kg selexipag compared with the vehicle-treated group, corresponding to the decrease in RVSP.

Fig 3

Hemodynamic effects of selexipag in the early stage of SuHx rats.

Effect of selexipag on right ventricular systolic pressure (RVSP) (A), the ratio of the weight of the right ventricle to that of the left ventricle + septum (RV/LV+S) (B), mean arterial blood pressure (MAP) (C) and heart rate (HR) (D). Statistical analyses were performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-3w, *P<0.05 and **P<0.01 vs. SuHx-6w. Values are means ± S.E.M. N = 8–10 per group.

Fig 4

Effect of selexipag on occlusive lesions in the early stage of SuHx rats.

Representative photographs of occlusive lesions of pulmonary vessels (scale bars = 20 μm) (A) and the percentage of occlusive lesions among the vessels (B). Statistical analysis was performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-3w, **P<0.01 vs. SuHx-6w. Values are means ± S.E.M. N = 8–10 per group.

Effects of selexipag on hemodynamic parameters, RV hypertrophy and obstructive pulmonary vascular remodeling in the late stage of SuHx rats

For the experiment on the early-pathology phase, selexipag at 30 mg/kg, but not 10 mg/kg, significantly improved RVSP and occlusive lesions. Therefore, in the next step, we used selexipag at 30 mg/kg to investigate the late-pathology phase. For the experiment on the late-pathology phase, SuHx rats were maintained under normoxic conditions for two weeks after exposure to 10% oxygen and then administered selexipag or vehicle for three weeks. Both RVSP and RV/(LV+S) were significantly elevated at the start of treatment (SuHx 5w; from 37.9 ± 0.8 to 116.2 ± 5.1 mmHg for RVSP and from 0.23 ± 0.01 to 0.62 ± 0.03 for RV/(LV+S)) and showed no further increase in the vehicle-treated group (SuHx 8w; 114.6 ± 7.3 mmHg for RVSP and 0.63 ± 0.03 for RV/(LV+S)) (Fig 5A and 5B). In the selexipag-treated group, RVSP (82.1 ± 8.1 mmHg) and RV/(LV+S) (0.43 ± 0.02) were significantly decreased not only compared with the vehicle-treated group (SuHx 8w) but also compared with the pre-treatment group (SuHx 5w). No effect on MAP or HR was observed (Fig 5C and 5D). Similar to the elevation of RVSP, the rate of occlusive lesions had significantly increased to 41 ± 5% five weeks after Sugen 5416 injection in the pre-treatment group (SuHx 5w) and showed no further significant change at eight weeks in the vehicle-treated group (SuHx 8w; 54 ± 5%) (Fig 6A and 6B). The rate of occlusive lesions was significantly reduced to 28 ± 5% in the group treated with 30 mg/kg selexipag not only compared with the vehicle-treated group but also compared with the pre-treatment group. An elevation of RVSP propagates an elevation of pulmonary artery pressure and leads to thickening of the medial wall of the pulmonary arteries in PAH patients [28]. The medial walls of pulmonary arteries were thickened in SuHx rats (normoxia, 12.2 ± 1.1%; SuHx 5w, 27.4 ± 1.7%; SuHx 8w, 29.5 ± 1.6%) and the thickening was significantly attenuated in the selexipag-treated group (16 ± 1.1%) compared with both the pre-treatment group and the vehicle-treated group (Fig 6A and 6C).

Fig 5

Hemodynamic effects of selexipag in the late stage of SuHx rats.

RVSP (A), RV/LV+S (B), mean arterial blood pressure (MAP) (C) and heart rate (HR) (D). Statistical analyses were performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-5w, **P<0.01 vs. SuHx-8w. Values are means ± S.E.M. N = 10 per group.

Fig 6

Effect of selexipag on occlusive lesions in the late stage of SuHx rats.

Representative photographs of occlusive lesions (top; scale bar = 20 μm) and medial wall thickness of pulmonary vessels (bottom; scale bars = 50 μm) (A), the percentage of occlusive lesions among the vessels (B) and the percentage of medial wall thickness (C). Statistical analyses were performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-5w, **P<0.01 vs. SuHx-8w. Values are means ± S.E.M. N = 10 per group.

Antiproliferative and apoptosis-inducing effect of selexipag in occluded small lung vessels from the late stage in SuHx rats

To investigate the detailed mechanism of the protective action of selexipag on PAH, we used lung vessels obtained from the late-pathology phase, because we presumed that it reflected the clinical setting in which selexipag is administered after establishment of the disease. Vascular occlusive lesions in the lungs of PAH patients are consistent with abnormal cell proliferation and vessel fibrosis [28,29]. An increase in cells positive for the proliferating cell marker Ki-67 and the expression of fibrotic proteins, including collagen type I, are observed in occluded lung vessels of SuHx rats [10,20]. The proportion of lung vessels with Ki-67-positive cells was 41 ± 2.9% in the pre-treatment group (SuHx 5w), a value that was already significantly increased over normoxia (16 ± 1.7%), and it progressed to 65 ± 1.7% in the vehicle-treated group (SuHx 8w) (Fig 7A and 7B). The selexipag-treated group showed a significant decrease to 29 ± 3.0% compared with both the vehicle-treated and the pre-treatment group. Ki-67-positive cells were observed in the luminal layer or neointima of occluded small vessels, but rarely in vessels of normal appearance. To investigate the lineage of the Ki-67-positive cells, we performed immunostaining with antibody against von Willebrand factor (vWF), a marker of endothelial cells, and carefully selected the same vessels which harbored Ki-67 positive cells. vWF was stained in the monolayered intima but not in the media or adventitia in the non-occluded and partially occluded vessels, whereas Ki-67-positive cells were observed in both the monolayered intima and the media in the partially and completely occluded vessels. We also assessed cell apoptosis in the occluded vessels by TUNEL staining, a method for identifying apoptotic cells by detecting DNA fragmentation. TUNEL-positive cells were more apparent in the intima-media complex of the occluded lung vessels of the selexipag-treated group than in the vehicle-treated group (Fig 7C).

Fig 7

Effect of selexipag on cell proliferation and apoptosis in occlusive lesions in the late stage.

Cells positive for the cell proliferation marker Ki-67 and terminal deoxynucleotidyl transferase-mediated 2'-deoxyuridine 5'-triphosphate nick-end labeling (TUNEL), a method for identifying apoptotic cells, were detected immunohistochemically (arrows). Representative photomicrographs of vessels from each group (top, Ki-67; bottom, von Willebrand factor; scale bars = 20 μm) (A), percentage of Ki-67-positive vessels (B) and representative photomicrographs of vessels with TUNEL-positive cells (C). Statistical analysis was performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-5w, **P<0.01 vs. SuHx-8w. Values are means ± S.E.M. N = 10 per group.

Effect of selexipag on fibrosis in small lung vessels in the late stage of SuHx rats

We immunohistologically assessed the expression of collagen type I, an extracellular matrix protein associated with fibrosis, in lung vessels. As with the proportion of occluded lung vessels, the signal intensity of anti-collagen type I antibody was significantly increased in the pre-treatment group (SuHx 5w; 1.06 ± 0.04 pixels/μm2; normoxia, 0.49 ± 0.03 pixels/μm2), with no further change in the vehicle-treated group (SuHx 8w; 1.04 ± 0.05 pixels/μm2) (Fig 8). The selexipag-treated group showed a significant reduction in signal intensity to 0.81 ± 0.04 pixels/μm2 compared with both the vehicle-treated and the pre-treatment group.

Fig 8

Effect of selexipag on expression of collagen type I in the late stage.

Collagen type I was detected immunohistochemically. Representative photomicrographs of vessels from each group (scale bar = 20 μm) (A) and the number of positive pixels per square micrometer of vessel area (B). Statistical analysis was performed using Tukey’s test. ##P<0.01 vs. normoxia, ††P<0.01 vs. SuHx-5w, **P<0.01 vs. SuHx-8w. Values are means ± S.E.M. N = 10 per group.

Effect of selexipag on mortality and RV hypertrophy in an SuHx-induced Fischer rat model of PAH

To assess the beneficial effect of selexipag on mortality by RV failure in PAH, we administered selexipag or vehicle to Fischer rats with SuHx-induced PAH for 21 days after the end of exposure to hypoxia and recorded the mortality of each group (N = 10 per group). The first death in the vehicle-treated group occurred on day 34 after Sugen 5416 injection, and the survival rate was 30% on day 42 (Fig 9A). Seven rats in the vehicle-treated group died of right heart failure. All animals in the selexipag-treated group survived until day 42, and the mortality of the selexipag-treated group was significantly lower than that of the vehicle-treated group (P<0.01; log-rank test). We sacrificed all surviving animals on day 42 to measure their RV hypertrophy. The RV hypertrophy was significantly improved in the selexipag-treated group compared with the vehicle-treated group (vehicle, 0.62 ± 0.03; selexipag, 0.50 ± 0.02; P<0.05; Student’s t-test; Fig 9B).

Fig 9

Effect of selexipag on mortality and RV hypertrophy in Fischer rats with SuHx-induced PAH.

Kaplan-Meier survival curves for the two groups (A) and RV/LV+S in the surviving animals on day 42 (B). Statistical analysis was performed using log-rank analysis for survival and Student’s t-test for RV/LV+S. *P<0.05 vs. vehicle. N = 10 per group.

Discussion

In this study, we demonstrate the pharmacological effect of selexipag on the pathological features of PAH in the SD SuHx rat model and on mortality by RV failure in the Fischer SuHx rat model. A three-week treatment with selexipag significantly improved not only RV hypertrophy and hemodynamic parameters such as RVSP but also occlusive vascular remodeling in both the early and the late stage in SD SuHx rats. Furthermore, selexipag also attenuated the mortality rate of Fischer SuHx rats.

We have previously reported that an active metabolite of selexipag, MRE-269, has potent vasodilator effects not only on strips of lung vessels dissected from healthy humans, pigs and rats, but also on remodeled lung vessels dissected from rats with monocrotaline (MCT)-induced PAH [17,30]. This suggests that selexipag has the potential to induce vasodilation in lung vessels not only in normal rats but also in PAH model rats. However, the acute vasodilator effects of selexipag on RVSP and homeostatic systemic blood pressure have not yet been investigated in an in vivo model. The elevation of RVSP in a U46619-induced acute PAH model mimics the increase in vascular tone in the pathology of PAH. Our results show that selexipag effectively vasodilated the lung vessels and decreased RVSP with no effect on systemic blood pressure. Consistent with the rapid hydrolysis of selexipag to MRE-269 and the long half-life of MRE-269 in rats [31], the vasodilator effect of selexipag was stably maintained for at least 2 h, the last time point of the experiments. This long-lasting pulmonary vasodilation should have a favorable effect on the pathology of PAH, and the small effect on the systemic resistance vessels suggests a low risk of hypotension as an adverse effect.

To investigate the pharmacological effects of selexipag on the phenotypes of PAH in the SuHx model, we designed two protocols for the time course of treatment. The time course of development of the PAH phenotype in the SD SuHx model varies slightly among reports [10,32]. In our experiments, SD SuHx rats showed significant increases in RVSP and the proportion of occlusive lesions observed three weeks after Sugen 5416 injection, and both were further increased at six weeks. So we decided on treatment with selexipag from three to six weeks as the early-stage protocol to assess its suppressive effects on the progressive disease and from five to eight weeks as the late-stage protocol to assess its therapeutic effects on the established disease. Selexipag significantly improved the increases in RVSP, RV hypertrophy and occlusive vascular remodeling in both the early and the late stage. Moreover, selexipag also showed improvements compared with the pre-treatment group in the late stage. The medial wall thickness of pulmonary arteries was also attenuated by selexipag treatment in the late stage. These results suggest that selexipag has beneficial effects on not only the hemodynamics but also on occlusive vascular remodeling in established PAH pathology.

Selexipag significantly improved the hemodynamics and RV hypertrophy at doses of 20–60 mg/kg/day in SD SuHx rats and 6 mg/kg/day in MCT-induced PAH rats, whereas the approved clinical dose for patients with PAH is up to 3.2 mg/day. The area under plasma concentration–time curve from 0 to infinity (AUC0–∞) of MRE-269 after oral administration of 60 mg/kg/day selexipag in rats is estimated to be about 100-fold higher than the AUC0–∞ of MRE-269 after oral administration of 3.2 mg selexipag in healthy humans [6,33]. The difference in the dose setting of selexipag between the rat model and human patients is partly owing to the species differences in the binding affinity of selexipag for the prostacyclin receptor. Thus, the binding affinity of MRE-269 for the human prostacyclin receptor is about 10-fold higher than for the rat prostacyclin receptor [27]. In addition, like selexipag, treprostinil improves the hemodynamics of an SuHx rat model at a dose over 10-fold higher than that used in the clinical or monocrotaline-induced PAH rat model [16]. The difference in the dose required to show effectiveness may be due to differences in the severity of PAH in the models.

The other prostacyclin receptor agonists iloprost and treprostinil have been assessed for their efficacy in SD SuHx rats. Thus, inhalation of an aerosol containing iloprost for two weeks improves RV function and partially reverses RV fibrosis in SuHx rats, but does not improve RV hypertrophy or the elevation of pulmonary vascular resistance [15]. On the other hand, continuous subcutaneous infusion of treprostinil with an osmotic pump for three weeks improves both hemodynamics and RV function in SD SuHx rats, but does not improve pulmonary vascular remodeling [16]. Although an echocardiographic analysis of the effects of selexipag on RV function and structure in animal models has not yet been reported, an increase in cardiac output was observed in PAH patients treated with selexipag in a clinical study [34]. This suggests that, as with iloprost and treprostinil, an improvement in RV function may have contributed to the improved hemodynamics observed in the selexipag-treated group in our study. In contrast to iloprost and treprostinil, selexipag improved occlusive vascular remodeling in SD SuHx rats. Hemodynamic unloading by pulmonary artery banding and reducing blood flow in the lungs reverses occlusive vascular lesions in the SuHx model [35]. This suggests that a strong reduction in hemodynamic stress by pulmonary vasodilation may improve pulmonary vascular remodeling. Of all prostacyclin receptor agonists tested, selexipag shows the most potent vasodilator effect on dissected rat small pulmonary artery, probably due to its high selectivity for the prostacyclin receptor and low binding affinity for other vasoconstrictive prostaglandin receptors [17,27]. Furthermore, selexipag does not induce activation of the β-arrestin pathway or desensitization of the prostacyclin receptor, and the vasodilator effect of selexipag on skin blood flow in rats is not attenuated by repeated administration [27,36]. It is probably because of these pharmacological features that selexipag decreased hemodynamic stress in our SuHx rats more effectively than did other prostacyclin receptor agonists.

Several groups have reported that alpha-smooth muscle actin (SMA)-positive smooth muscle cells are observed in both the neointima and the media in occlusive pulmonary vessels in SD SuHx rats. On the other hand, vWF-positive endothelial cells are observed in the monolayered intima, but not in the media [37-39]. In the selexipag-treated group, we observed Ki-67-positive cells not only in the neointima and media which occluded the small vessels but also in the monolayered intima which showed vWF staining. This suggests that the proliferation of both smooth-muscle-like cells in the neointima and media and endothelial-like cells in the monolayered intima were activated in the lung vessels of SuHx rats. It is in good agreement with the pathology of PAH patients that the abnormalities of both smooth muscle cells and endothelial cells are important in the development of vascular remodeling [40]. In the present study, consistent with the reduction in vascular occlusive lesions, the numbers of Ki-67-positive cells were decreased and TUNEL-positive apoptotic cells were observed in the intima-media complex in the occluded lung vessels in the selexipag-treated group. It seems likely that TUNEL staining was shown by smooth-muscle-like cells in both the neointima and the media. The antiproliferative or proapoptotic effect of selexipag on vascular smooth muscle cells or endothelial cells dissociated from SD SuHx rats has not been tested. However, prostacyclin receptor agonists, including selexipag, are known to have an antiproliferative effect on human pulmonary arterial smooth muscle cells stimulated by platelet-derived growth factor [36], whereas direct effects of selexipag on vascular endothelial cells and apoptosis have not yet been reported. The antiproliferative effect of selexipag on vascular smooth muscle cells may also contribute to the improvement of occlusive vascular remodeling. The detailed mechanism of the anti-vascular remodeling effects of selexipag needs further investigation.

Selexipag decreased collagen type I expression in the adventitia of lung vessels. In addition, in normal human lung fibroblasts, MRE-269 significantly inhibited the transforming growth factor (TGF) β1-stimulated production of procollagen type I C-peptide (S1 Fig). Another group has also reported that selexipag has antifibrotic effects on human fibroblasts stimulated by platelet-derived growth factor [41]. In addition to the potent pulmonary vasodilator effect of selexipag, its direct effects on vascular smooth muscle cells and fibroblasts may have contributed to its improvement of pulmonary vascular remodeling and fibrosis in SuHx rats.

Selexipag attenuated the development of RV hypertrophy and reduced the mortality associated with RV failure in SuHx rats. As far as we know, this is the first demonstration of a reduction in mortality risk in an SuHx rat model among drugs approved for PAH treatment. Increased afterload due to persistent high pulmonary arterial pressure causes RV hypertrophy and eventually leads to RV failure in patients with PAH [42,43]. The reduction in mortality obtained in our study probably resulted from a decrease in the afterload of the RV through pulmonary vasodilation with a resulting improvement in pulmonary vascular remodeling.

In conclusion, selexipag improved hemodynamics, occlusive vascular remodeling and mortality in SuHx-induced rat models of severe PAH. These effects were probably based on the potent prostacyclin receptor agonistic effect of selexipag on pulmonary vessels. This is the first report to show that activation of the prostacyclin receptor exerts therapeutic effects on not only the hemodynamics but also on occlusive vascular remodeling and mortality by RV failure in SuHx rat models. Selexipag has been approved for and is in use in the clinical treatment of PAH worldwide. It is likely that these beneficial effects on multiple aspects of PAH pathology contribute to the clinical outcomes in patients with PAH.

Supplemental materials and methods.

(DOCX)

Click here for additional data file.

Effect of MRE-269 on TGFβ-induced collagen production of normal human lung fibroblasts.

Statistical analyses were performed using Student’s t- test followed by Dunnett’s test. ##P<0.01 vs. TGFβ (-) group by Student’s t-test, **P<0.01 vs. TGFβ (+) group by Dunnett’s test. Values are means ± S.E.M. N = 4 per group.

(PPTX)

Click here for additional data file.

31 Jul 2020

PONE-D-20-17665

The selective PGI2 receptor agonist selexipag ameliorates Sugen 5416/hypoxia-induced pulmonary arterial hypertension in rats

PLOS ONE

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Reviewer #1: Comments to the authors:

Honda and colleagues have conducted a study of the effects of selexipag on rat models of pulmonary arterial hypertension. Authors used three animal models (the acute dose of U46619 and Sugen/Hypoxia on two rat strains, Sprague Dawley and Fischer). Honda et al. show the ability of selexipag to attenuate the vasoconstrictive effect of U46619. They also showed the decrease in RVSP and Fulton’s index in the SuHx Sprague Dawley rats (2 time-points), as well as the decreased mortality in Fischer rats. In the SuHx model Honda et al showed a decrease in KI-67 positive cells with an increase in apoptotic cells and a decrease in collagen deposition in the lung vasculature.

Major points:

1. The authors decided to use term developing-phase and established-phase of PAH when referring to the morphological changes in the lung, however the readership may interpret it as a development of PAH itself, which is not correct: In the previous, comprehensive study of the SuHx rat PAH model, Legchenko et al, (Sci Trans Med, 2018) show that at the 3+1 week (1 week post-weeks hypoxia hypoxia) animals do have an established PAH phenotype, and in the following 5 weeks right heart failure develops. Thus, I would recommend to call the stages the authors did their experiments in as “early” and “late-stage”.

2. While selexipag indeed significantly decreased the RVSP in SuHx rats, the pressures still remained suprasytemic (from 141 to 102 mmHg). This needs to be mentioned since basically the rats still have severe PAH: Most other groups find average RVSP of 90-110mmHg, and some groups found average RVSP of only 50-70mmmHg in SuHx rats. Bogaard HJ et al. AJRCCM, 2019, doi: 10.1164/rccm.201906-1200LE.

2. For the TUNEL assay it would be beneficial to have the aSMA staining (on the next serial section slide) to show the apoptotic cells are smooth muscle cells and not endothelial.

3. In the conclusion: lines 585-586: “…taken together with those obtained for SuHx rats in the present study, suggest that selexipag would have beneficial effects on multiple aspects of PAH pathology”.

The authors need to re-write these conclusions as selexipag has been approved (as authors write themselves in the introduction, line 69-70) and is widely used in clinical care for > 3 years.

5. Many cited references are outdated.

Critique by section

ABSTRACT:

Wording could be improved. Write Sprague Dawley strain and Fischer strain – so far only the Fischer strain is mentioned at the end of the abstract.

INTRODUCTION:

Exchange references, as listed below under REFERENCES.

METHODS/DISCUSSION:

The authors should discuss the straindependency of the phenotype of the SuHx rat model, with best results in the Sprague Dawley rats who lack a emphysema phenotype (whie WKY rats were reported by one group to have emphysema disqualifying the WKY rat SuHx as model for PAH):

References to be cited ad discussed:

The Adult Sprague-Dawley Sugen-Hypoxia Rat Is Still "the One:" A Model of Group 1 Pulmonary Hypertension: Reply to Le Cras and Abman.

Bogaard HJ, Legchenko E, Ackermann M, Kühnel MP, Jonigk DD, Chaudhary KR, Sun X, Stewart DJ, Hansmann G.

Emphysema Is-at the Most-Only a Mild Phenotype in the Sugen/Hypoxia Rat Model of Pulmonary Arterial Hypertension.

Bogaard HJ, Legchenko E, Chaudhary KR, Sun XQ, Stewart DJ, Hansmann G. Am J Respir Crit Care Med. 2019 Dec 1;200(11):1447-1450. doi: 10.1164/rccm.201906-1200LE.

Rational for choosing SuHx: The SuHx rat is considered superior vs. MCT and other rodent models by a group of experts: Bonnet S et al. Translating Research into Improved Patient Care in Pulmonary Arterial Hypertension, Am J Resp Crit Care Med, 2017. Also, while near every intrervention seems to work in the monocrotain /MCT) rat PAH model, this is clearly not the case in SuHx exposed rats that are rather treatment resistant.

The most comprehensive phenotyping study on the SUHx rat model, by means of echo, right and left heart cath, MRI and PET CT has been conducted by Legchenko et al. and this paper should be cited and discussed (see experimental design, RV assessment, PVD assessment):

PPARγ agonist pioglitazone reverses pulmonary hypertension and prevents right heart failure via fatty acid oxidation.

Legchenko E, Chouvarine P, Borchert P, Fernandez-Gonzalez A, Snay E, Meier M, Maegel L, Mitsialis SA, Rog-Zielinska EA, Kourembanas S, Jonigk D, Hansmann G. Sci Transl Med. 2018 Apr 25;10(438):eaao0303. doi: 10.1126/scitranslmed.aao0303.

Why did the authors use male Sprague Dawley rats (original ad standard strain) for most experiments but Fischer rats (male ?) for the survival experiment ?

Just let us know the rationale of change of protocol ?

What was the mean RVSP that the authors measured in Fischer rats (in SD, the authors report 130-140mmHg !! which is international record for the average RVSP in this SuHx model)

As reported by authors, Fischer and Sprague-Dawley rats developed similar increases in RVSP to 100 mm Hg.

See: Jiang B, Deng Y, Suen C, Taha M, Chaudhary KR, Courtman DW, et al.

Marked strain-specific differences in the SU5416 rat model of severe

pulmonary arterial hypertension. Am J Respir Cell Mol Biol 2016;54:

461–468.

It seems Selexipag was given s.c. twice daily subcutaneously, please write this clearly

So far: “Selexipag was suspended in 0.5% methylcellulose solution and 10 or 30 mg/kg of the compound was administered twice daily.”

This mmeas the total daily dosse of Selexipag was 20-60mg/rat/day while the current max. recommended adult patient dose is 1.6 mg (1600 mcg) twice daily PO. Please discuss in the discussion why such a super physiological dose was used.

Why did the authors not incorporate the drug into the food, especially in the reversal experiments, as described by Legchenko et al. Science Translational Medicine, 2018 ?

Both Sugen (SU5416) and selexipag was dissolved in 0.5% methylcellulose solution … to acieve a complete suspension or even clear solutiio with 0.5% methylcellulose is nearly impossible, and probably responsibe for the great variattey of RVSP reported and variance within the same study. DMSO as vehicle ad solvent would have been an alternative. Was the controls the authors used a vehicle control (0.5% methylcellulose), meaning where the rats not treated with selelcipag s.c. ttwice daily injected twiche daily with 0.5% methylcellulose vol../vol. ? Twice daily injections are quite stressful,a ddn if the authors have not done the control injections, this must be listed under limiitations of the study.

Systemic blood pressure was measured by tail cuff, which inferior to invasive measure of aortic and LV enddiastolic pressure. However, invasive SAP and LVEDDP was not different between the groups, as shown by Legchhenko et al. (no postcapillary PH). This observation should be cited, but then I think it is ok to have only the tail cuff data.

RESULTS:

Figure 1. What were the absolute changes in mmHg ? Pllease write that at least in the text. It is hard to crasp from the figure that showns only percentage changes.

Figure 1B: are the two last data points indeed not significant?

Figure 2. indicates that al rats received vehicle s.c. – good.

Figure 3. I suggest to use color coding for the different groups in stead of multipe difrenet b/w patterns. Same for the other figures with multiple columns.

In the authors’ ATS abstract from 2017, the RVSP in SuHx rats with PA was very high (ca- 130-140 mmHg), and could be significantly decreased to 100 mmHg, but only with the higher Selxipag dose of 30mg/kg/dose s.c. twice daily.

https://www.atsjournals.org/doi/abs/10.1164/ajrccm-conference.2017.195.1_MeetingAbstracts.A4221

The Novel and Selective Prostacyclin Receptor Agonist Selexipag Vasodilates Human Pulmonary Arteries in an Endothelium-Independent Manner and Ameliorates Pulmonary Arterial Hypertension in Rat Models

Kazuya Kuramoto , Chiaki Fuchikami , Keiji Kosugi , Yohei Honda , Michiko Oka , . American Journal of Respiratory and Critical Care Medicine 2017;195:A4221

Please make sure that all authors of this ATS abstract are also listed as authors on this original article

Kazuya Kuramoto , Chiaki Fuchikami , Keiji Kosugi , Yohei Honda , Michiko Oka , Keiichi Kuwano

i.e., should Michiko Oka not be an author on the paper ? If not, please provide the freasonig why this has been changed after an abstract with the name of this author as been published.

Figure 3. What is known about the effect of selexipag on RV systolic and diastolic function in SuHx exposed rats ? The authors should provide the (blinded) RVEDP measurements, RV dp/dt max and min. It would be greta if the authors had echo data but it seems it would require the entire study if they do not have those data.

Figure 4A, 6A, 8A – include the name of the staining in the figure (as in Figure 7).

Figure 6B – 1st column can’t be empty as you performed statistics on it.

Figure 7. As far as I know, it was not know that selecipag ca induce apoptosis in the media (TUNEL assay), probably SMC. Can this not simpy explained by the supraphysiological doses ? Can the authors provide cell culture experimental data that underpins this finding with much lower selexipag’s active metabolite in culture, in the dose range of 10nM – 1uM ?

Overall, I feel the figures could be also in panels so that the total number of figures can be reduced to approx.. 7, but leave this to the discretion of the handling editor.

Have the authors performed any expression studies on the RV and LV, or even only whole lung, to get a handle on the RNA/protein changes induced by selexipag and its active metabolite…. ?

Have the authors performed any expression studies on other organs, such as liver and idney that are probably also affected by the demonstrated systemic to suprasystemic RV and PA pressures in SuHx rats ?

Minor points:

1. In the introduction after approval (lines 69-70), it is important to address the use of selexigag in pediatric patients (Geerdink et al, Pulm Circ 2017; Koo et al., Cardiol Young, 2019; Rothman et al, Pulm Circ 2020; Hansmann et al, J H Lung Transplant 2020).

2. There is data not included in the figues in the discussion (lines 532-536). Please, include in the figures/supplemental figures.

3. Discussion line 568: “Selexipag partially reversed RV hypertrophy” – you haven’t shown a direct effect of selexipag on the heart or isolated cardiomyocytes, also as was shown in Legchenko et al.,Sci Trans Med, 2018, at 3+1 weeks, RV diameter is not changed in Sprague Dawley rats, so at the time of administration of selexipag, the RV was not hypertrophied. Needs to be re-written as: administration of selexipag attenuated the development of RV hypertrophy.

Reviewer #2: Honda et al. investigated the effects of selexipag in sugen/hypoxia induced PAH. Selexipag has acute vasodilator effect and improved RVSP, RVH and pulmonary artery remodeling in prevention and treatment protocol. This study is interested; however, some concerns are included.

1. What caused the reverse pulmonary artery remodeling? Could reduction of shear stress by vasodilation of selexipag reverse pulmonary artery remodeling? Or Did selexipag directly affect pulmonary arteries?

2. Authors should emphasize the novelty of this study. Similar studies had already published.

3. Did authors examine the proapoptotic and antiproliferative effects in pulmonary artery endothelial or smooth muscle cells from sugen/hypoxia rats?

4. How many dose of selexipag 30mg/kg is in human setting?

**********

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2 Sep 2020

Prof. Dr. Michael Bader

Academic Editor

PLOS ONE

Dear Dr. Bader:

PLOS ONE PONE-D-20-17665

The selective PGI2 receptor agonist selexipag ameliorates Sugen 5416/hypoxia-induced pulmonary arterial hypertension in rats

Thank you very much for your e-mail of August 1st 2020, and for the review of our manuscript.

I hope that you’ll find my response to the comments satisfactory. I am re-submitting a revised manuscript for consideration of publication in PLOS ONE.

Thank you very much for your consideration.

Sincerely yours,

Yohei Honda

Enclosures:

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(Ans.) We have ensured that our manuscript meets PLOS ONE's style requirements.

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(2) Please describe the post-operative care received by the animals, including the frequency of monitoring and the specific clinical, physiological and behavioural criteria used to assess animal health and well-being.

(Ans.) We have rewritten the appropriate parts of the Methods section according to your suggestion.

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[All authors are employed by Nippon Shinyaku Co., Ltd.].

(Ans.) We have already stated it in the cover letter.

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[All authors are employed by Nippon Shinyaku Co., Ltd.].

(Ans.) We have already stated it in the cover letter.

Reviewers' comments:

We deeply appreciate your kind advice on our manuscript. Bearing your comments in mind, we have revised the manuscript to make our conclusions more convincing. Below we reply to the specific points you raised.

Reviewer #1: Comments to the authors:

Honda and colleagues have conducted a study of the effects of selexipag on rat models of pulmonary arterial hypertension. Authors used three animal models (the acute dose of U46619 and Sugen/Hypoxia on two rat strains, Sprague Dawley and Fischer). Honda et al. show the ability of selexipag to attenuate the vasoconstrictive effect of U46619. They also showed the decrease in RVSP and Fulton’s index in the SuHx Sprague Dawley rats (2 time-points), as well as the decreased mortality in Fischer rats. In the SuHx model Honda et al showed a decrease in KI-67 positive cells with an increase in apoptotic cells and a decrease in collagen deposition in the lung vasculature.

Major points:

1. The authors decided to use term developing-phase and established-phase of PAH when referring to the morphological changes in the lung, however the readership may interpret it as a development of PAH itself, which is not correct: In the previous, comprehensive study of the SuHx rat PAH model, Legchenko et al, (Sci Trans Med, 2018) show that at the 3+1 week (1 week post-weeks hypoxia hypoxia) animals do have an established PAH phenotype, and in the following 5 weeks right heart failure develops. Thus, I would recommend to call the stages the authors did their experiments in as “early” and “late-stage”.

(Ans.) According your suggestion, we changed the description of the experimental stages to “early” and “late-stage”.

2. While selexipag indeed significantly decreased the RVSP in SuHx rats, the pressures still remained suprasytemic (from 141 to 102 mmHg). This needs to be mentioned since basically the rats still have severe PAH: Most other groups find average RVSP of 90-110mmHg, and some groups found average RVSP of only 50-70mmmHg in SuHx rats. Bogaard HJ et al. AJRCCM, 2019, doi: 10.1164/rccm.201906-1200LE.

(Ans.) In the revised manuscript, we state that rats in the selexipag-treated group had still had high RVSP. We had calibrated the measurement system with a mercury manometer before each experiment and this is now mentioned in the Methods section (lines 201-203).

2. For the TUNEL assay it would be beneficial to have the aSMA staining (on the next serial section slide) to show the apoptotic cells are smooth muscle cells and not endothelial.

(Ans.) We did not perform immunohistochemical staining with anti-alpha-SMA antibody. However, several groups have reported that, like the pathology of PAH patients, alpha-SMA-positive smooth muscle cells are observed in both neointima and media in occlusive pulmonary vessels in SD SuHx rats. On the other hand, vWF-positive endothelial cells are observed in monolayered intima, but not in media [Tamura et al., 2018, Jernigan et al., 2017, Mair et al., 2014]. In our study, TUNEL-positive cells were observed in both neointima and media; this suggests that at least some of the TUNEL-positive cells would be smooth muscle cells. We have added a description of these previous reports and references to the discussion section (lines 581-585).

Tamura Y, Phan C, Tu L, Hiress M, Thuillet R, Jutant EM et al.

Ectopic upregulation of membrane-bound IL6R drives vascular remodeling in pulmonary arterial hypertension

Clin Invest. 2018;128(5):1956-1970. doi: 10.1172/JCI96462

Mair K, Wright A, Duggan N, Rowlands D, Hussey M, Roberts S et al.

Sex-dependent influence of endogenous estrogen in pulmonary hypertension

Am J Respir Crit Care Med. 2014;190(4):456-67. doi: 10.1164/rccm.201403-0483OC

Jernigan N, Naik J, Weise-Cross L, Detweiler N, Herbert L, Yellowhair T, Resta T

Contribution of reactive oxygen species to the pathogenesis of pulmonary arterial hypertension

PLoS One. 2017;12(6):e0180455. doi: 10.1371/journal.pone.0180455

3. In the conclusion: lines 585-586: “…taken together with those obtained for SuHx rats in the present study, suggest that selexipag would have beneficial effects on multiple aspects of PAH pathology”.

The authors need to re-write these conclusions as selexipag has been approved (as authors write themselves in the introduction, line 69-70) and is widely used in clinical care for > 3 years.

(Ans.) According your suggestion, we mentioned that selexipag has been approved in Discussion and Abstract section (lines 632-633 and lines 51-52).

5. Many cited references are outdated.

(Ans.) According your suggestion, we updated references.

Critique by section

＊ABSTRACT:

Wording could be improved. Write Sprague Dawley strain and Fischer strain – so far only the Fischer strain is mentioned at the end of the abstract.

(Ans.) We have improved the wording as you suggest (line 40).

＊INTRODUCTION:

Exchange references, as listed below under REFERENCES.

(Ans.) According your suggestion, we updated references.

＊METHODS/DISCUSSION:

The authors should discuss the straindependency of the phenotype of the SuHx rat model, with best results in the Sprague Dawley rats who lack a emphysema phenotype (whie WKY rats were reported by one group to have emphysema disqualifying the WKY rat SuHx as model for PAH):

References to be cited ad discussed:

The Adult Sprague-Dawley Sugen-Hypoxia Rat Is Still "the One:" A Model of Group 1 Pulmonary Hypertension: Reply to Le Cras and Abman.

Bogaard HJ, Legchenko E, Ackermann M, Kühnel MP, Jonigk DD, Chaudhary KR, Sun X, Stewart DJ, Hansmann G.

Emphysema Is-at the Most-Only a Mild Phenotype in the Sugen/Hypoxia Rat Model of Pulmonary Arterial Hypertension.

Bogaard HJ, Legchenko E, Chaudhary KR, Sun XQ, Stewart DJ, Hansmann G. Am J Respir Crit Care Med. 2019 Dec 1;200(11):1447-1450. doi: 10.1164/rccm.201906-1200LE.

Rational for choosing SuHx: The SuHx rat is considered superior vs. MCT and other rodent models by a group of experts: Bonnet S et al. Translating Research into Improved Patient Care in Pulmonary Arterial Hypertension, Am J Resp Crit Care Med, 2017. Also, while near every intrervention seems to work in the monocrotain /MCT) rat PAH model, this is clearly not the case in SuHx exposed rats that are rather treatment resistant.

The most comprehensive phenotyping study on the SUHx rat model, by means of echo, right and left heart cath, MRI and PET CT has been conducted by Legchenko et al. and this paper should be cited and discussed (see experimental design, RV assessment, PVD assessment):

PPARγ agonist pioglitazone reverses pulmonary hypertension and prevents right heart failure via fatty acid oxidation.

Legchenko E, Chouvarine P, Borchert P, Fernandez-Gonzalez A, Snay E, Meier M, Maegel L, Mitsialis SA, Rog-Zielinska EA, Kourembanas S, Jonigk D, Hansmann G. Sci Transl Med. 2018 Apr 25;10(438):eaao0303. doi: 10.1126/scitranslmed.aao0303.

(Ans.) We have added a description of SuHx rat models and references you kindly mentioned to the introduction section (lines 82-98).

＊Why did the authors use male Sprague Dawley rats (original ad standard strain) for most experiments but Fischer rats (male ?) for the survival experiment ?

＊Just let us know the rationale of change of protocol ?

(Ans.) To date, most studies of the SuHx model have used Sprague-Dawley rats, so in most experiments we used Sprague-Dawley rats. They show a severe PAH phenotype, but show excellent survival for up to 14 weeks. To evaluate the effect of selexipag on mortality, we used Fischer rats, which exhibit very high mortality because of strain-dependent differences [Suen CM et al., 2019]. We added this point in the introduction section (lines 82-98).

Suen CM, Chaudhary KR, Deng Y, Jiang B, Stewart DJ.

Fischer rats exhibit maladaptive structural and molecular right ventricular remodelling in severe pulmonary hypertension: A genetically prone model for right heart failure.

Cardiovasc Res. 2019;115: 788–799. doi:10.1093/cvr/cvy258

What was the mean RVSP that the authors measured in Fischer rats (in SD, the authors report 130-140mmHg !! which is international record for the average RVSP in this SuHx model)

As reported by authors, Fischer and Sprague-Dawley rats developed similar increases in RVSP to 100 mm Hg.

See: Jiang B, Deng Y, Suen C, Taha M, Chaudhary KR, Courtman DW, et al.

Marked strain-specific differences in the SU5416 rat model of severe

pulmonary arterial hypertension. Am J Respir Cell Mol Biol 2016;54:

461–468.

(Ans.) Because we have not measured RVSP in the survival study with Fischer SuHx rats, we do not have any data to compare with the RVSP of SD SuHx rats. On the other hand, the development of RV hypertrophy of Fischer SuHx rats (RV/(LV+S); 0.62 ± 0.03, Fig. 9B) was quite similar to that of SD SuHx rats (RV/(LV+S); 0.63 ± 0.02, Fig. 3B). This is in good agreement with the reference you mention, which shows that Fischer SuHx rats develop RV hypertrophy and increased RVSP at the same level as SD SuHx rats. Therefore, we guess that the RVSP of our Fischer SuHx rats would probably be increased to levels similar to those of SD SuHx rats.

＊It seems Selexipag was given s.c. twice daily subcutaneously, please write this clearly

So far: “Selexipag was suspended in 0.5% methylcellulose solution and 10 or 30 mg/kg of the compound was administered twice daily.”

(Ans.) Selexipag was given orally twice daily. This is now clearly stated in the revised Materials and Methods (lines 168-171).

＊This mmeas the total daily dosse of Selexipag was 20-60mg/rat/day while the current max. recommended adult patient dose is 1.6 mg (1600 mcg) twice daily PO. Please discuss in the discussion why such a super physiological dose was used.

(Ans.) We have added an explanation of this point in an extra paragraph in the Discussion (lines 537-552).

＊Why did the authors not incorporate the drug into the food, especially in the reversal experiments, as described by Legchenko et al. Science Translational Medicine, 2018 ?

(Ans.) Mixing selexipag into the food affected the amount of food intake of the SD SuHx rats. In order to ensure that every rat was given the same dose of selexipag, we administered selexipag to rats orally.

＊Both Sugen (SU5416) and selexipag was dissolved in 0.5% methylcellulose solution … to acieve a complete suspension or even clear solutiio with 0.5% methylcellulose is nearly impossible, and probably responsibe for the great variattey of RVSP reported and variance within the same study. DMSO as vehicle ad solvent would have been an alternative. Was the controls the authors used a vehicle control (0.5% methylcellulose), meaning where the rats not treated with selelcipag s.c. ttwice daily injected twiche daily with 0.5% methylcellulose vol../vol. ? Twice daily injections are quite stressful,a ddn if the authors have not done the control injections, this must be listed under limiitations of the study.

(Ans.) Sugen was suspended in buffer containing 0.5% carboxymethyl cellulose sodium salt, 0.9% NaCl, 0.4% polysorbate 80 and 0.9% benzyl alcohol, and a single injection was given subcutaneously to induce PAH. On the other hand, selexipag was dissolved in 0.5% methylcellulose solution, and administered orally twice daily. Vehicle control rats were administered 0.5% methylcellulose solution orally twice daily instead of selexipag solution. We revised the materials and methods section to clarify the route of administration of these materials (lines 160-171).

＊Systemic blood pressure was measured by tail cuff, which inferior to invasive measure of aortic and LV enddiastolic pressure. However, invasive SAP and LVEDDP was not different between the groups, as shown by Legchhenko et al. (no postcapillary PH). This observation should be cited, but then I think it is ok to have only the tail cuff data.

(Ans) In the U46619-induced acute PAH rat model, we measured mean arterial pressure (MAP) by inserting a cannula into the right femoral artery. This is now clearly stated in the methods section (lines 138-139). The MAP measured invasively by cannula and noninvasively by tail cuff were not very different in our experiments. So we chose the noninvasive tail cuff method to measure MAP in later experiments.

RESULTS:

＊Figure 1. What were the absolute changes in mmHg ? Pllease write that at least in the text. It is hard to crasp from the figure that showns only percentage changes.

(Ans.) We have added the absolute changes in mmHg to the Results (line 300).

＊Figure 1B: are the two last data points indeed not significant?

(Ans.) According to the statistical analysis, there was no significant difference between the two groups in mean arterial pressure.

＊Figure 2. indicates that al rats received vehicle s.c. – good.

(Ans.) Vehicle control rats received 0.5% methylcellulose solution orally. We added this point in an extra paragraph in the Materials and Methods (lines 168-169).

＊Figure 3. I suggest to use color coding for the different groups in stead of multipe difrenet b/w patterns. Same for the other figures with multiple columns.

(Ans.) We changed to color coding of figures.

＊In the authors’ ATS abstract from 2017, the RVSP in SuHx rats with PA was very high (ca- 130-140 mmHg), and could be significantly decreased to 100 mmHg, but only with the higher Selxipag dose of 30mg/kg/dose s.c. twice daily.

https://www.atsjournals.org/doi/abs/10.1164/ajrccm-conference.2017.195.1_MeetingAbstracts.A4221

The Novel and Selective Prostacyclin Receptor Agonist Selexipag Vasodilates Human Pulmonary Arteries in an Endothelium-Independent Manner and Ameliorates Pulmonary Arterial Hypertension in Rat Models

Kazuya Kuramoto , Chiaki Fuchikami , Keiji Kosugi , Yohei Honda , Michiko Oka , . American Journal of Respiratory and Critical Care Medicine 2017;195:A4221

Please make sure that all authors of this ATS abstract are also listed as authors on this original article

Kazuya Kuramoto , Chiaki Fuchikami , Keiji Kosugi , Yohei Honda , Michiko Oka , Keiichi Kuwano

i.e., should Michiko Oka not be an author on the paper ? If not, please provide the freasonig why this has been changed after an abstract with the name of this author as been published.

(Ans.) The figure of RVSP and RV hypertrophy in the ATS abstract A4221 is exactly same as Fig. 3A and 3B in this article. The administration of 30 mg/kg selexipag p.o. twice daily significantly decreased RVSP. Michiko Oka contributed to the ex vivo experiments to assess the vasodilator effect of selexipag on dissected pulmonary arterial vessel rings in the poster. The work about the vasodilator effect of selexipag has been already published as indicated below. Therefore, she is not included as an author in this study.

Fuchikami C, Murakami K, Tajima K, Homan J, Kosugi K, Kuramoto K, Oka M, Kuwano K.

A comparison of vasodilation mode among selexipag (NS-304; [2-{4-[(5,6- diphenylpyrazin-2-yl)(isopropyl)amino]butoxy}-N-(methylsulfonyl) acetamide]), its active metabolite MRE-269 and various prostacyclin receptor agonists in rat, porcine and human pulmonary arteries.

Eur J Pharmacol. 2017;795: 75–83. doi:10.1016/j.ejphar.2016.11.05

＊Figure 3. What is known about the effect of selexipag on RV systolic and diastolic function in SuHx exposed rats ? The authors should provide the (blinded) RVEDP measurements, RV dp/dt max and min. It would be greta if the authors had echo data but it seems it would require the entire study if they do not have those data.

(Ans.) We have not measured the effect of selexipag on RV systolic and diastolic function, because we do not have any diagnostic machines such as ultrasound echocardiography or MRI. We agree that the effect of selexipag on RV systolic and diastolic function should be investigated in a future study.

＊Figure 4A, 6A, 8A – include the name of the staining in the figure (as in Figure 7).

(Ans.) We have added the name of the staining in the figure.

＊Figure 6B – 1st column can’t be empty as you performed statistics on it.

(Ans.) As previously reported by Shinohara et al., occlusive lesions were not observed in pulmonary vessels in normal rats. Thus, the percentages of all normal group were 0%. We performed statistical analysis between the normal group (0%) and the SuHx groups.

Shinohara T, Sawada H, Otsuki S, Yodoya N, Kato T, Ohashi H, et al.

Macitentan reverses early obstructive pulmonary vasculopathy in rats: early intervention in overcoming the survivin-mediated resistance to apoptosis.

Am J Physiol Lung Cell Mol Physiol. 2015;308: L523-38. doi:10.1152/ajplung.00129.2014

＊Figure 7. As far as I know, it was not know that selecipag ca induce apoptosis in the media (TUNEL assay), probably SMC. Can this not simpy explained by the supraphysiological doses ? Can the authors provide cell culture experimental data that underpins this finding with much lower selexipag’s active metabolite in culture, in the dose range of 10nM – 1uM ?

(Ans.) We have not assessed the proapoptotic and antiproliferative effect of selexipag and its active metabolite, MRE-269, on pulmonary artery endothelial cells or smooth muscle cells from SuHx rats. However, hemodynamic unloading by pulmonary artery banding and reducing blood flow in the lungs reverses occlusive vascular lesions in the SuHx model [Abe et al., 2016]. This suggests that a strong reduction in hemodynamic stress by pulmonary vasodilation may improve pulmonary vascular remodeling. We mention this in the revised discussion section (lines 567-571).

Abe K, Shinoda M, Tanaka M, Kuwabara Y, Yoshida K, Hirooka Y, et al.

Haemodynamic unloading reverses occlusive vascular lesions in severe pulmonary hypertension. Cardiovasc Res. 2016;111: 16–25. doi:10.1093/cvr/cvw070

＊Overall, I feel the figures could be also in panels so that the total number of figures can be reduced to approx.. 7, but leave this to the discretion of the handling editor.

(Ans.) According to submission guidelines, the maximum height of the figures is 2625 pixels at 300 dpi. So we separated the results of hemodynamics and vascular remodeling in SuHx rats into two figures in order to keep the resolution of the images and make them easy to see. However, we will follow the instructions of editor about the figures.

＊Have the authors performed any expression studies on the RV and LV, or even only whole lung, to get a handle on the RNA/protein changes induced by selexipag and its active metabolite…. ?

＊Have the authors performed any expression studies on other organs, such as liver and idney that are probably also affected by the demonstrated systemic to suprasystemic RV and PA pressures in SuHx rats ?

(Ans.) We have not measured the effect of selexipag on RNA or protein expression in lung or other organs. We agree that the effect of selexipag on RNA or protein expression should be investigated in a future study.

Minor points:

1. In the introduction after approval (lines 69-70), it is important to address the use of selexigag in pediatric patients (Geerdink et al, Pulm Circ 2017; Koo et al., Cardiol Young, 2019; Rothman et al, Pulm Circ 2020; Hansmann et al, J H Lung Transplant 2020).

(Ans.) We totally agree that the beneficial effect of selexipag in pediatric PAH patients has been reported by several groups, and it is an important observation to improve the treatment of pediatric patients. However, at present, selexipag has not been approved for the treatment of pediatric PAH patients in any country. We are concerned that describing the use of selexipag in pediatric patients could mislead readers into thinking that selexipag is also approved or recommended for the treatment of pediatric patients. This is an ethical issue because Nippon Shinyaku Co. Ltd., which we are employed by, is the manufacturer of selexipag. Therefore, we are afraid that we should not address the use of selexipag in pediatric patients in this paper.

2. There is data not included in the figues in the discussion (lines 532-536). Please, include in the figures/supplemental figures.

(Ans.) We added this data as supplemental figure 1.

3. Discussion line 568: “Selexipag partially reversed RV hypertrophy” – you haven’t shown a direct effect of selexipag on the heart or isolated cardiomyocytes, also as was shown in Legchenko et al.,Sci Trans Med, 2018, at 3+1 weeks, RV diameter is not changed in Sprague Dawley rats, so at the time of administration of selexipag, the RV was not hypertrophied. Needs to be re-written as: administration of selexipag attenuated the development of RV hypertrophy.

(Ans.) According to your suggestion, we have rewritten the sentence in the discussion section (line 617).

Reviewer #2: Honda et al. investigated the effects of selexipag in sugen/hypoxia induced PAH. Selexipag has acute vasodilator effect and improved RVSP, RVH and pulmonary artery remodeling in prevention and treatment protocol. This study is interested; however, some concerns are included.

We deeply appreciate your kind advice on our manuscript. Accepting your criticism, the manuscript was revised to make our conclusions more convincing. Hereafter, we will reply to the points you raised.

1. What caused the reverse pulmonary artery remodeling? Could reduction of shear stress by vasodilation of selexipag reverse pulmonary artery remodeling? Or Did selexipag directly affect pulmonary arteries?

(Ans.) Selexipag is a potent prostacyclin receptor agonist and it significantly reduced RVSP in the U46619-induced acute PAH rat model. Thus, the reduction of shear stress by the vasodilator effect of selexipag is probably the primary mechanism of action of the improvement of pulmonary artery remodeling. The antiproliferative effect of selexipag on occlusive vessels in SuHx rats has not been clearly elucidated yet, but it may contribute to the improvement of vascular remodeling. We added a sentence about this point in the discussion section (lines 604-605).

2. Authors should emphasize the novelty of this study. Similar studies had already published.

(Ans.) This is the first report to show that activation of the prostacyclin receptor exerts therapeutic effects on not only the hemodynamics but also on occlusive vascular remodeling and mortality by RV failure in SuHx rat models. We added a description of our findings in abstract/introduction/discussion sections to emphasize the novelty of this study (lines 50-55, 99-109 and 626-635).

3. Did authors examine the proapoptotic and antiproliferative effects in pulmonary artery endothelial or smooth muscle cells from sugen/hypoxia rats?

(Ans.) We have not assessed the proapoptotic and antiproliferative effect of selexipag on pulmonary artery endothelial cells or smooth muscle cells from SuHx rats. We agree that this point should be investigated in a future study.

4. How many dose of selexipag 30mg/kg is in human setting?

(Ans.) Selexipag significantly improved the hemodynamics and RV hypertrophy at doses of 20–60 mg/kg/day in SD SuHx rats, whereas the approved clinical dose for patients with PAH is up to 3.2 mg/day. The AUC0–∞ of MRE-269 after oral administration of 60 mg/kg/day selexipag in rats is about 100-fold higher than the AUC0–∞ of MRE-269 after oral administration of 3.2 mg selexipag in healthy humans [Asaki et al., 2015, Kaufman et al., 2015]. The difference in the dose setting of selexipag between the rat model and human patients is partly because of the species differences in the pharmacokinetics and the binding affinity of selexipag for the prostacyclin receptor. Thus, the binding affinity of MRE-269 for the human prostacyclin receptor is about 10-fold higher than for the rat prostacyclin receptor [Kuwano et al., 2007]. In addition, treprostinil improves the hemodynamics of an SuHx rat model at a dose over 10-fold higher than that used in the clinical or monocrotaline-induced PAH rat model [Chaudhary et al., 2018]. The difference in the dose required to show effectiveness may be due to differences in the severity of PAH in the models. We added mention of these points in an extra paragraph in the discussion section (lines 537-552).

Submitted filename: Letter_resubmission.docx

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1 Oct 2020

The selective PGI2 receptor agonist selexipag ameliorates Sugen 5416/hypoxia-induced pulmonary arterial hypertension in rats

PONE-D-20-17665R1

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5 Oct 2020

PONE-D-20-17665R1

The selective PGI2 receptor agonist selexipag ameliorates Sugen 5416/hypoxia-induced pulmonary arterial hypertension in rats

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