Model Difference in the Effect of Cilostazol on the Development of Experimental Pulmonary Hypertension in Rats

Model Difference in the Effect of Cilostazol on the Development of Experimental Pulmonary Hypertension in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research article Model Difference in the Effect of Cilostazol on the Development of Experimental Pulmonary Hypertension in Rats Toshikazu Ito, Erquan Zhang, Ayaka Omori, Jane Kabwe, Masako Kawai, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-228016/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2021 Read the published version in BMC Pulmonary Medicine → Version 1 posted 10 You are reading this latest preprint version Abstract Background: Preventing pulmonary vascular remodeling is a key strategy for pulmonary hypertension (PH). Causes of PH include pulmonary vasoconstriction and inflammation. This study aimed to determine whether cilostazol (CLZ), a phosphodiesterase-3 inhibitor, prevents monocrotaline (MCT)- and chronic hypoxia (CH)-induced PH development in rats. Methods: Fifty-one male Sprague-Dawley rats were fed rat chow with (0.3% CLZ) or without CLZ for 21 days after a single injection of MCT (60 mg/kg) or saline. Forty-eight rats were fed rat chow with and without CLZ for 14 days under ambient or hypobaric (air at 380 mmHg) CH exposure. Mean PAP (mPAP), the right ventricle weight-to-left ventricle+septum weight ratio (RV/LV+S), percentages of muscularized peripheral pulmonary arteries (%Muscularization) and medial wall thickness of small muscular arteries (%MWT) were assessed. Protein expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (peNOS), AKT, pAKT and IκB in lung tissue was measured by Western blotting. Monocyte chemotactic protein (MCP)-1 mRNA in lung tissue was also assessed. Results: mPAP [35.1±1.7 mmHg (MCT) (n=9) vs.16.6±0.7 (control) (n=9) (p<0.05); 29.1±1.5 mmHg (CH) (n=10) vs. 17.5±0.5 (control) (n=10) (p<0.05)], RV/LV+S [0.40±0.01 (MCT) (n=18) vs. 0.24±0.01 (control) (n=10) (p<0.05); 0.41±0.03 (CH) (n=13) vs. 0.27±0.06 (control) (n=10) (p<0.05)], and %Muscularization and %MWT were increased by MCT injection and CH exposure. CLZ significantly attenuated these changes in the MCT model [mPAP 25.1±1.1 mmHg (n=11) (p<0.05), RV/LV+S 0.30±0.01 (n=14) (p<0.05)]. In contrast, these CLZ effects were not observed in the CH model. Lung eNOS protein expression was unchanged in the MCT model and high in the CH model. Lung protein expression of AKT, phosphorylated AKT, and IκB was downregulated by MCT, which was attenuated by CLZ; the CH model did not change these proteins. Lung MCP-1 mRNA levels were increased in MCT rats but not CH rats. Conclusion: We found model differences in the effect of CLZ on PH development. CLZ might have a preventable effect on PH development in an inflammatory PH model but not in a vascular structural change model of PH preceded by vasoconstriction. Thus, the preventive effect of CLZ on PH development might be dependent on PH etiology. Pulmonology monocrotaline chronic hypoxia cilostazol pulmonary hypertension nitric oxide Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Background Pulmonary hypertension (PH) is characterized by an increase in pulmonary artery pressure (PAP), right ventricular hypertrophy (RVH), and functional and/or structural vascular changes [ 1 , 2 ]. Possible causes of PH include pulmonary vasoconstriction, diffuse micro-thromboembolism, and pulmonary vascular remodeling [ 1 , 2 , 3 ]. In all conditions causing PH in humans [ 3 , 4 , 5 ] and experimental models [ 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 ], vascular changes include new muscularization of normally nonmuscular peripheral pulmonary arteries and medial hypertrophy of muscular arteries. PH may be encountered in the intensive care unit in patients with acute respiratory distress syndrome (ARDS) [ 17 , 18 , 19 ], congenital heart disease with left-to-right shunt [ 3 , 20 , 21 ], mitral valve disease [ 22 ], and interstitial pulmonary fibrosis [ 23 ], as well as after cardiothoracic surgery [ 24 , 25 ]. In all conditions causing PH, including ARDS in patients [ 4 , 5 ] and experimental rat models such as monocrotaline (MCT)-induced PH [ 6 , 12 , 13 , 26 , 27 , 28 , 29 , 36 ] and chronic hypoxia-induced PH [ 6 , 7 , 8 , 9 , 30 , 31 ], vascular remodeling involves new muscularization of normally nonmuscular peripheral pulmonary arteries and medial hypertrophy of muscular arteries. A total of 7.46% of critically ill patients admitted to the intensive care unit met the ARDS criteria. The prevalence of PH in these ARDS patients was as high as 46.6% [ 32 ]. Nitric oxide (NO) is a vasodilator and suppressor of smooth muscle cell proliferation [ 1 , 2 ], and the bioavailability of NO is reduced in patients with PH and in experimental PH models [ 33 , 34 , 35 ]. We and others have shown that modulation to increase NO production ameliorates the development of PH and vascular remodeling [ 6 , 7 , 36 ]. Cilostazol (CLZ) is a selective phosphodiesterase-3 inhibitor that increases intracellular cyclic AMP, which inhibits platelet aggregation and induces peripheral vasodilation. The antiplatelet agent CLZ is indicated in intermittent claudication in peripheral arterial disease [ 37 ], thrombotic complications of coronary angioplasty [ 38 ] and secondary stroke prevention [ 39 ]. Through cAMP-dependent and cAMP-independent mechanisms, CLZ also causes phosphorylation of eNOS, which increases NO production in the aortas of diabetic rats [ 40 ], human aortic endothelial cells [ 41 ], and rat cultured smooth muscle cells [ 42 ]. CLZ also has anti-inflammatory effects [ 43 , 44 , 45 ]. Through activating the NO synthase-NO pathway or preventing inflammatory responses, CLZ might prevent the development of PH, as suggested in an earlier study [ 46 ] in which PH was not fatal. Since the pathogenesis and severity of PH are heterogeneous [ 1 , 2 , 3 , 47 ], we determined the effect of CLZ on the development of two established experimental models of PH: MCT-induced PH rats [ 6 , 12 , 13 , 14 , 15 , 36 , 46 , 48 , 49 , 50 , 51 , 52 , 53 ] and chronic hypoxia (CH)-induced PH rats [ 6 , 7 , 8 , 9 , 10 , 11 , 34 , 35 , 54 , 55 ]. The MCT-induced PH rats in this study were used as experimental fatal PH. Methods 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 [56] 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. Animal groups MCT21 model 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. CH model 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]. MCT28 model 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-P R , 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. Survival experiment 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. Data analysis 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 StatView5.0 R . Differences were considered significant at P<0.05. Results Body weight MCT21 model All rats gained body weight steadily. MCT rats had significantly lower body weights than saline control rats from day 4 to the last day of the experiment. CLZ had no effects on body weight gain in either the MCT or saline control rats (Figure 3A). CH model CH rats lost weight during the first several days of hypoxia exposure but regained weight afterward. The air rats gained weight steadily. After the start of hypoxia exposure, CH rats showed significantly lower body weights than air rats. CLZ treatment had no effect on the body weight of either air or CH rats (Figure 3B). Dosage of CLZ MCT model Approximate dosages (mg) of CLZ were calculated by the equation food intake (g) x 0.003 x 1000, which is the average dosage per kg per day throughout the experimental course. The dosage of CLZ was ~230 mg/kg/day in the Sal/CLZ+ group and ~200 mg/kg/day in the MCT/CLZ+ group (Figure 4A). Although the dosage decreased to 64 mg/kg/day on the day of MCT injection, the dosage was similar in the Sal/CLZ+ and MCT/CLZ+ groups from day 4 throughout the experiment. CH model The dosage of CLZ was ~250 mg/kg/day in the air/CLZ+ group and ~220 mg/kg/day in the CH/CLZ+ group (Figure 4B). Although the dosage decreased to 11 mg/kg/day on the first day of hypoxia exposure, the dosage was similar in the Air/CLZ+ and CH/CLZ+ groups from day 4 throughout the experiment. mPAP and RV/LV+S, mPAP/mAP and sRVP MCT21 model Comparing the effects of MCT administration between Sal21/CLZ- and MCT21/CLZ-, mPAP [16.56±0.73 (n=9) vs. 35.33±1.67 (n=9) mmHg (p<0.05) (Fig 5A)], mPAP/mAP [0.16±0.01 (n=8) vs. 0.38±0.01 (n=8) (P<0.05) (Fig 5B)], and RV/LV+S [0.24±0.01 (n=10) vs. 0.40±0.01 (n=18) (P<0.05) (Fig 5C)] were all significantly higher in MCT21/CLZ-, suggesting that MCT caused PH and RVH. Comparing the effects of CLZ treatment between MCT21/CLZ+ and MCT21/CLZ-, mPAP [25.09±1.06 (n=11) (P<0.05)], mPAP/mAP [0.25±0.01 (n=11)], and RV/LV+S [0.30±0.01 (n=14)] in MCT21/CLZ+ were significantly lower than those in MCT21/CLZ- (Fig. 5A, C, E), suggesting that CLZ treatment ameliorated the development of PH and RVH. MCT28 model Comparing the effects of MCT administration between Sal28/CLZ- and MCT28/CLZ-, sRVP and RV/LV+S was significantly higher in MCT28/CLZ-: sRVP, 30.20±3.35 (n=3) vs. 76.63±3.20 (n=8) mmHg (p<0.05) (Fig 6A); RV/LV+S, 0.28±0.02 (n=5) vs. 0.63±0.04 (n=8) (P<0.05) (Fig 6B). Comparing the effects of CLZ treatment between MCT28/CLZ+ and MCT28/CLZ-, sRVP [35.28±1.90 (n=8) (P<0.05) (Fig 6A)] and RV/LV+S [0.32±0.04 (n=9) (P<0.05)] in MCT28/CLZ+ were significantly lower than those in MCT28/CLZ- (Fig. 6B), suggesting again that CLZ treatment ameliorated the development of PH and RVH. CH model CH caused PH with a mPAP of 29.1±1.5 mmHg (n=10) (CH/CLZ-) compared to that in Air/CLZ- 17.5±0.5 mmHg (n=10) (p<0.05). There were no significant differences between CH/CLZ+ and CH/CLZ- individuals (Figure 5B). RV/LV+S was higher in CH/CLZ- 0.41±0.03 (n=13) than in Air/CLZ- 0.27±0.06 (n=10) (p<0.05). There were no significant differences in RV/LV+S between CH/CLZ+ and CH/CLZ- (Figure 5F). The ratio of mPAP/mAP was also higher in CH/CLZ- than Air/CLZ-, with no significant difference between CH/CLZ+ and CH/CLZ- (Figure 5D). Survival CLZ treatment significantly improved survival from 26 days after MCT injection in MCT-induced PH rats (Figure 7). Vascular structural changes MCT21 model The %Muscularization was higher in those with external diameters between 15 and 50 µm (Figure 8A) and those between 51 and 100 µm (Figure 8B) in MCT21/CLZ- than in Sal21/CLZ-. MCT21/CLZ+ had significantly lower %Muscularization in both sizes of arteries than MCT21/CLZ- (Figure 8A, B). The %MWT in MCT21/CLZ- was higher than that in Sal21/CLZ- in the small muscular arteries between 51 and 100 µm in external diameter at the alveolar duct level, and those between 101 and 200 µm in external diameter were usually accompanied by terminal or respiratory bronchioles (Figure 8C, D). MCT21/CLZ+ had a significantly lower %MWT than MCT21/CLZ- (Figure 8C, D). CH model The %Muscularization (Figure 9A, B) and %MWT (Figure 9C, D) were significantly higher in CH/CLZ- than Air/CLZ-, whereas CH/CLZ+ had no significant differences compared to CH/CLZ- (Figure 9A, B, C, D). Hematocrit Hematocrit was similar among the Sal21/CLZ-, Sal21/CLZ+, MCT21/CLZ- and MCT21/CLZ+ groups: 43.9±0.7% (n=9), 41.4±1.4% (n=9), 41.3±1.5% (n=16), and 42.4±1.5% (n=14), respectively. Hematocrit in CH/CLZ- 56.1±1.6% (n=9) was significantly higher than in Air/CLZ- 42.8±1.7% (n=9). CH/CLZ+ 56.1±1.6% (n=11) had values similar to those of CH/CLZ-. Western blotting and PCR eNOS and peNOS MCT had no effect on eNOS protein expression (Figure 10A, B), whereas CH upregulated eNOS protein expression (Figure 10C). MCT also had no effect on eNOS mRNA levels (Figure 10G, H), whereas chronic hypoxia increased eNOS mRNA expression (Figure 10I). MCT decreased peNOS protein expression (Figure 10D, E), whereas CH did not (Figure 10F). CLZ increased eNOS mRNA levels in the MCT21 group (Figure 10G) and decreased peNOS expression in the CH group (Figure 10F). AKT and pAKT MCT significantly downregulated AKT protein expression in the MCT21 model (Figure 11A) and pAKT protein expression in the MCT28 model (Figure 11E). CLZ significantly attenuated this decrease in pAKT protein expression in the MCT28 model (Figure 11E) and increased AKT mRNA expression in the MCT28 model (Figure 11H). In the CH model, AKT mRNA, AKT protein and pAKT expression were unchanged, and CLZ had no effect (Figure 11C, F, I). IκB and HMGB-1 MCT downregulated IκB (Figure 12A, B) and HMGB-1 (Figure 12D, E) protein expression in both the MCT21 and MCT28 models (Figure 12A, B, D, E). CLZ significantly attenuated the downregulation of both IκB (Figure 12B) and HMGB-1 (Figure 12E) protein expression in the MCT28 model. In the CH model, IκB and HMGB-1 protein expression was unchanged, and CLZ had no effect (Figure 12C, F). MCP-1 The mRNA expression of MCP-1 was higher in MCT21/CLZ- than in Sal21/CLZ-. There was no significant difference between MCT21/CLZ- and MCT21/CLZ+ (Figure 13A). In the MCT28 and CH models, there were no differences in MCP-1 mRNA levels among the groups (Figure 13B, C). Discussion The MCT and CH models were used to determine the model difference in the effect of CLZ on the development of PH. In both the MCT model and CH model, rats developed increases in mPAP and RV/LV + S, suggesting the successful development of PH. CLZ treatment ameliorated the development of MCT-induced PH. Hypertensive pulmonary vascular remodeling (i.e., new muscularization of peripheral pulmonary arteries and medial hypertrophy of muscular arteries) in the MCT model was ameliorated by CLZ treatment. In the CH model, CLZ treatment did not ameliorate the development of PH or hypertensive vascular remodeling. High hematocrit contributes to high PAP in the CH model in addition to hypertensive pulmonary vascular remodeling [ 11 ], where the decrease in hematocrit is expected to reduce PAP. Since the hematocrit in both the CH and MCT models was not changed by CLZ, attenuation of the mPAP by CLZ treatment was not due to the decrease in hematocrit. Medial hypertrophy of the muscular artery indicates the hypertrophy and hyperplasia of vascular smooth muscle cells, whereas new muscularization of normally nonmuscular arteries indicates the differentiation of pericytes to mature smooth muscle cells [ 11 , 12 ]. Previous studies showed that increasing endogenous NO could ameliorate these structural changes [ 6 , 36 ]. Earlier studies revealed that eNOS mRNA is increased in the lungs of CH-induced PH rats [ 54 , 57 ], consistent with the present results. Since CLZ increases NOS expression in cultured endothelium [ 40 , 41 ], we expected to observe the upregulation of NOS due to CLZ in CH lung tissue. CLZ might have no effect on NOS synthesis, at least in CH whole lung tissue, since eNOS protein levels in the lung tissue were similar between CH-induced PH rats with and without CLZ. This suggests that combined CH and CLZ treatment did not further enhance eNOS expression compared with CH-induced NOS upregulation. Although eNOS mRNA expression was increased by CLZ in the MCT21 model, which is consistent with an earlier study [ 46 ], CLZ had no effect on the protein expression of eNOS and peNOS in either model. Translation of eNOS mRNA might be impaired in the MCT model; therefore, it is difficult to explain the preventable effect of CLZ in the MCT model by increased NO production. MCT-induced PH rats [ 6 , 12 , 13 , 14 , 15 , 36 , 46 , 48 , 49 , 50 , 51 , 52 , 53 ] have been used to investigate pulmonary vascular remodeling in inflammatory-related PH, including ARDS [ 4 , 5 ]. CH-induced PH [ 6 , 7 , 8 , 9 , 10 , 11 , 34 , 35 , 54 , 55 ] is a type of PH due to hypoxia that includes patients residing at high altitude and patients with chronic obstructive pulmonary disease. Endothelial injury precedes the increase in PAP in MCT-induced PH [ 51 ], whereas the increase in PAP precedes the development of vascular changes in CH-induced PH [ 10 ]. A previous study showed that chronic NO inhalation prevented the development of PH and pulmonary vascular remodeling in CH-induced PH [ 7 ] but not in MCT-induced PH [ 12 ]. NO inhalation causes selective pulmonary vasodilation. Thus, the different effects of inhaled NO between PH models suggest that reversing vasoconstriction is effective in preventing the development of PH in some forms in which vasoconstriction is the initial insult. Since CLZ could not prevent the development of CH-induced PH, we speculate that CLZ has a less potent pulmonary vasodilating effect. Endothelial injury is the initial insult in MCT-induced PH [ 51 ]. CLZ has been reported to promote endothelial regeneration in injured carotid arteries [ 58 ] and endothelial proliferation in lymphatics [ 59 ]. There are several limitations in this study. First, the present results were in adult male rats, and we must be cautious in discussing neonatal and juvenile rats and/or infant and pediatric human patients, since age and sex influence pulmonary hypertension in chronic hypoxia [ 60 ], and animal models do not completely recapture human disease [ 16 ]. Second, the dosage of CLZ was higher in the present study than in the previous study [ 46 ]. We used this concentration because chow including 0.3% CLZ was used in spontaneously hypertensive rats [ 56 ]. The rats in the previous study [ 46 ] did not have a lethal condition compared with the rats in the present study. Third, this study is observational and not mechanistic. CLZ enhances Akt phosphorylation in human aortic endothelial cells [ 41 ] and in neuroblastoma cells [ 43 ]. In human aortic endothelial cells [ 41 ] and canine coronary blood vessels [ 61 ], PI3/AKT-dependent NO production has been reported. Since attenuation of the decrease in pAKT, an active form of AKT, was detected in the MCT model with no changes in the CH model, this effect of CLZ might partly explain the model difference in the effect of CLZ. Although one of the targets of pAKT is eNOS [ 61 ], this is not the case in the present study because we could not detect upregulation of peNOS expression. An earlier study in our laboratory showed that MCT treatment significantly reduced the protein levels of IκBα in lung tissues, which was restored by pyrrolidine dithiocarbamate (PDTC), an NFκB inhibitor [ 48 ]. NFκB is a transcription factor that regulates the transcription of genes involved in inflammatory responses. The decrease in the protein levels of IκBα has been reported to be associated with the upregulation of NFκB activity [ 62 ]. IκBα was unchanged in CH-induced PH rats but decreased in MCT-induced PH rats in the current study, consistent with our earlier study [ 48 ], suggesting that inflammatory components play a greater role in the etiology of MCT than in the chronic hypoxia model. Increased mRNA expression of lung MCP-1 in the present study also supports the inflammatory component of the MCT model. The mechanism and role of MCP-1 in the inflammatory response are chemotactic and activating effects on monocytes/macrophages [ 53 ]. In the MCT model, we and others have shown macrophage infiltration into the alveolar wall by 14 days after MCT injection [ 15 , 48 ]. The plasma and bronchoalveolar lavage fluid (BALF) MCP-1 levels increased transiently and then returned to normal levels [ 53 ]. We also showed that lung MCP-1 mRNA was elevated in MCT21 rats in the present study and showed increases in BALF MCP-1 and TNFα levels measured by ELISA in a previous study [ 14 ], suggesting the presence of inflammation in MCT rats. The anti-inflammatory effect of CLZ [ 43 , 63 ] might explain the reversal of decreased levels of IκBα in the lungs of MCT-injected rats. CLZ has been reported to inactivate NFκB [ 64 , 65 ]. Moreover, retinoic acid prevented the development of MCT-induced PH with the inhibition of MMP-1 [ 50 ]. NFκB induces the promotion of MMP-1 [ 66 ], and CLZ was reported to prevent MMP-1 in a cell study [ 67 ]. HMGB-1 is normally present as a nuclear protein and is passively released from damaged cells [ 44 ]. The lower expression of HMGB-1 in the lung tissue of MCT-injected rats might be due to the increased release of HMGB-1 protein into the circulation, which might reflect damage to the cells. A recent study showed that the increase in serum HMGB-1 is associated with the concurrent decrease in tissue HMGB-1 protein expression [ 68 ]. CLZ restored HMGB-1 protein expression to the control level 28 days after MCT injection, which might show an ability of CLZ to ameliorate cell damage and improve survival. Furthermore, CLZ inhibits HMGB-1 release in lipopolysaccharide-activated HMGB-1 release and increases the survival of endotoxemic mice [ 63 ]. In summary, the administration of CLZ prevented the development of PH in MCT-induced PH, although CH-induced PH development was not prevented by CLZ. The inhibitory effect of CLZ on the development of PH might depend on the etiology of PH, in which alterations in lung AKT, pAKT, and IκB might partly be related. Abbreviations PH pulmonary hypertension CLZ cilostazol MCT monocrotaline CH chronic (hypobaric) hypoxia RV/LV + S ratio of cardiac right ventricle/(left ventricle to septum) mPAP mean pulmonary artery pressure mAP mean artery pressure sRVP systolic right ventricular pressure PCR polymerase chain reaction %Muscularization percentages of muscularized arteries %MWT percent medial wall thickness eNOS endothelial nitric oxide synthase AKT serine-threonine protein kinase MCP-1 monocyte chemotactic protein-1 Declarations Ethics approval and consent to participate The Animal Care and Use Committee of Mie University School of Medicine approved the research protocol (No. 20-34 and 20-35). Consent for publication Not applicable Availability of data and materials All data generated or analyzed during this study are included in this published article [and its supplementary information files]. Competing interests The authors declare that they have no competing interests. Funding This work was financially supported by grants in aid for scientific research from the Japanese Ministry of Education, Science and Culture. (Grants-in-Aid 17K11075). Authors’ contributions TI and EZ contributed equally to this work, collecting and analyzing the data, and drafting the manuscript. AO, JK, JM, MK, and AO made substantial contributions to data acquisition and reviewing the manuscript. HS, AY, and YM contributed to the study design, statistical analysis, interpretation of data, and final approval of the manuscript. KM conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript. Competing interests We have no financial relationships to disclose. Acknowledgment We would like to thank Otsuka Pharmaceutical Co. for the gift of rat chow. References Archer SL, Weir EK, Wilkins MR. Basic science of pulmonary arterial hypertension for clinicians: new concepts and experimental therapies. 2010; 121:2045-66 Rabinovitch M. Molecular pathogenesis of pulmonary arterial hypertension. J Clin Invest. 2008; 118:2372-9. Rabinovitch M, Keane JF, Norwood WI, Castaneda AR, Reid L. Vascular structure in lung tissue obtained at biopsy correlated with pulmonary hemodynamic findings after repair of congenital heart defects. 1984; 69:655-67 Snow RL, Davies P, Pontoppidan H, Zapol WM, Reid L. Pulmonary vascular remodeling in adult respiratory distress syndrome. Am Rev Respir Dis. 1982;126:887-92 Tomashefski JF Jr, Davies P, Boggis C, Greene R, Zapol WM, Reid LM. The pulmonary vascular lesions of the adult respiratory distress syndrome. Am J Pathol. 1983; 112:112-26. Mitani Y, Maruyama K, Sakurai M. Prolonged administration of L-arginine ameliorates chronic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation.1997;96:689-97. Kouyoumdjian C, Adnot S, Levame M, Eddahibi S, Bousbaa H, Raffestin B. Continuous inhalation of nitric oxide protects against development of pulmonary hypertension in chronically hypoxic rats. J Clin Invest. 1994; 94:578-84. Zhang E, Jiang B, Yokochi A, Maruyama J, Mitani Y, Ma N, Maruyama K.Effect of all-trans-retinoic acid on the development of chronic hypoxia-induced pulmonary hypertension. Circ J. 2010; 74:1696-703 Zhang E, Maruyama J, Yokochi A, Mitani Y, Sawada H, Nishikawa M, Ma N, Maruyama K. Sarpogrelate hydrochloride, a serotonin 5HT2A receptor antagonist, ameliorates the development of chronic hypoxic pulmonary hypertension in rats. J Anesth. 2015; 29:715-23. Rabinovitch M, Gamble W, Nadas AS, Miettinen OS, Reid L. Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features. Am J Physiol. 1979;236:H818-27 Maruyama K, Ye CL, Woo M, Venkatacharya H, Lines LD, Silver MM, Rabinovitch M. Chronic hypoxic pulmonary hypertension in rats and increased elastolytic activity. Am J Physiol. 1991;261:H1716-26. Maruyama J, Maruyama K, Mitani Y, Kitabatake M, Yamauchi T, Miyasaka K. Continuous low-dose NO inhalation does not prevent monocrotaline-induced pulmonary hypertension in rats. Am J Physiol. 1997;272:H517-24. Ilkiw R, Todorovich-Hunter L, Maruyama K, J Shin, and M Rabinovitch. SC-39026, a serine elastase inhibitor, prevents muscularization of peripheral arteries, suggesting a mechanism of monocrotaline-induced pulmonary hypertension in rats. Circ Res 1989; 64: 814-25 Yamada Y, Maruyama J, Zhang E, Okada A, Yokochi A, Sawada H, Mitani Y, Hayashi T, Suzuki K, Maruyama K. Effect of thrombomodulin on the development of monocrotaline-induced pulmonary hypertension. J Anesth. 2014;28:26-33. Meyrick B, Gamble W, Reid L. Development of Crotalaria pulmonary hypertension: hemodynamic and structural study. Am J Physiol. 1980;239:H692-702. Stenmark KR, Meyrick B, Galie N, Mooi WJ, McMurtry IF. Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure. Am J Physiol Lung Cell Mol Physiol. 2009;297:L1013-32. Zapol WM, Snider MT. Pulmonary hypertension in severe acute respiratory failure. N Engl J Med. 1977; 296:476-80. Maruyama K, Nakai Y, Takeuchi M, Mizumoto T, Chikusa H, Muneyuki M. Verapamil reduced pulmonary hypertension in adult respiratory distress syndrome. J Anesth. 1994; 8:480-1. Maruyama K, Takeuchi M, Chikusa H, Muneyuki M. Reduction of intrapulmonary shunt by low-dose inhaled nitric oxide in a patient with late-stage respiratory distress associated with paraquat poisoning. Intensive Care Med. 1995;21:778-9. Maruyama K, Maruyama J, Utsunomiya H, Furuhashi K, Kurobuchi M, Katayama Y, Yada I, Muneyuki M. Effect of nicardipine on pulmonary hypertension after repair of congenital heart defects in early postoperative period. J Anesth. 1993; 7:95-101. Shimpo H, Mitani Y, Tanaka J, Mizumoto T, Onoda K, Tani K, Yuasa H, Yada I, Maruyama K. Inhaled low-dose nitric oxide for postoperative care in patients with congenital heart defects. Artif Organs. 1997; 21:10-3. Breitling S, Ravindran K, Goldenberg NM, Kuebler WM. The pathophysiology of pulmonary hypertension in left heart disease. Am J Physiol Lung Cell Mol Physiol. 2015;309:L924-41. Maruyama K, Kobayasi H, Taguchi O, Chikusa H, Muneyuki M. Higher doses of inhaled nitric oxide might be less effective in improving oxygenation in a patient with interstitial pulmonary fibrosis. Anesth Analg. 1995; 81:210-1. Zamanian RT, Haddad F, Doyle RL, Weinacker AB. Management strategies for patients with pulmonary hypertension in the intensive care unit. Crit Care Med. 2007;35:2037-50. Goldenberg NM, Rabinovitch M, Steinberg BE. Inflammatory Basis of Pulmonary Arterial Hypertension: Implications for Perioperative and Critical Care Medicine. Anesthesiology. 2019; 131:898-907. Hill NS, Warburton RR, Pietras L, Klinger JR. Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats. J Appl Physiol. 1997;83:1209-15. Schermuly RT, Kreisselmeier KP, Ghofrani HA, Yilmaz H, Butrous G, Ermert L, Ermert M, Weissmann N, Rose F, Guenther A, Walmrath D, Seeger W, Grimminger F. Chronic sildenafil treatment inhibits monocrotaline-induced pulmonary hypertension in rats. Am J Respir Crit Care Med. 2004;169:39-45. Dony E, Lai YJ, Dumitrascu R, Pullamsetti SS, Savai R, Ghofrani HA, Weissmann N, Schudt C, Flockerzi D, Seeger W, Grimminger F, Schermuly RT. Partial reversal of experimental pulmonary hypertension by phosphodiesterase-3/4 inhibition. Eur Respir J. 2008;31:599-610. Schermuly RT, Kreisselmeier KP, Ghofrani HA, Samidurai A, Pullamsetti S, Weissmann N, Schudt C, Ermert L, Seeger W, Grimminger F. Antiremodeling effects of iloprost and the dual-selective phosphodiesterase 3/4 inhibitor tolafentrine in chronic experimental pulmonary hypertension. Circ Res. 2004;94:1101-8. Eddahibi S, Raffestin B, Clozel M, Levame M, Adnot S. Protection from pulmonary hypertension with an orally active endothelin receptor antagonist in hypoxic rats. Am J Physiol. 1995;268:H828-35. Zhao L, Mason NA, Morrell NW, Kojonazarov B, Sadykov A, Maripov A, Mirrakhimov MM, Aldashev A, Wilkins MR. Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation. 2001; 104:424-8. Ñamendys-Silva S.A., Santos-Martínez L.E., Pulido T. Pulmonary hypertension due to acute respiratory distress syndrome. Braz J Med Biol Res. 2014; 47: 904–910. Klinger JR, Kadowitz PJ．The Nitric Oxide Pathway in Pulmonary Vascular Disease. Am J Cardiol. 2017; 120: S71-9. Maruyama J, Jiang BH, Maruyama K, Takata M, Miyasaka K. Prolonged nitric oxide inhalation during recovery from chronic hypoxia does not decrease nitric oxide-dependent relaxation in pulmonary arteries. 2004;126:1919-25. Maruyama J, Maruyama K. Impaired nitric oxide-dependent responses and their recovery in hypertensive pulmonary arteries of rats. Am J Physiol. 1994;266:H2476-88. Zhao YD, Courtman DW, Deng Y, Kugathasan L, Zhang Q, Stewart DJ. Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease. Circ Res. 2005; 96:442-50. Hiatt WR. The US experience with cilostazol in treating intermittent claudication. Suppl. 2005; 6:21-31. Tsuchikane E, Fukuhara A, Kobayashi T, Kirino M, Yamasaki K, Kobayashi T, Izumi M, Otsuji S, Tateyama H, Sakurai M, Awata N. Impact of cilostazol on restenosis after percutaneous coronary balloon angioplasty. 1999; 100:21-6 Noma K, Higashi Y. Cilostazol for treatment of cerebral infarction. Expert Opin Pharmacother. 2018; 19:1719-26. Suzuki K, Uchida K, Nakanishi N, Hattori Y. Cilostazol activates AMP-activated protein kinase and restores endothelial function in diabetes. Am J Hypertens. 2008; 21:451-7. Hashimoto A, Miyakoda G, Hirose Y, Mori T. Activation of endothelial nitric oxide synthase by cilostazol via a cAMP/protein kinase A- and phosphatidylinositol 3-kinase/Akt-dependent mechanism. Atherosclerosis. 2006; 189:350-7. Ikeda U, Ikeda M, Kano S, Kanbe T, Shimada K. Effect of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide production by vascular smooth muscle cells. Eur J Pharmacol. 1996; 314:197-202. Hassan M, Ibrahim MA, Hafez HM, Mohamed MZ, Zenhom NM, Abd Elghany HM. Role of Nrf2/HO-1 and PI3K/Akt Genes in the Hepatoprotective Effect of Cilostazol. Curr Clin Pharmacol. 2019; 14:61-7. Andersson U, Tracey KJ. HMGB1 is a therapeutic target for sterile inflammation and infection. Annu Rev Immunol. 2011; 29:139-62. Sakamoto T, Ohashi W, Tomita K, Hattori K, Matsuda N, Hattori Y. Anti-inflammatory properties of cilostazol: Its interruption of DNA binding activity of NF-κB from the Toll-like receptor signaling pathways. Int Immunopharmacol. 2018;62:120-31. Chang LT, Sun CK, Sheu JJ, Chiang CH, Youssef AA, Lee FY, Wu CJ, Yip HK. Cilostazol therapy attenuates monocrotaline-induced pulmonary arterial hypertension in rat model. Circ J. 2008; 72:825-31. Simonneau G, Gatzoulis MA, Adatia I, Celermajer D, Denton C, Ghofrani A, Gomez Sanchez MA, Krishna Kumar R, Landzberg M, Machado RF, Olschewski H, Robbins IM, Souza R. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol. 2013; 62: D34-41. Sawada H, Mitani Y, Maruyama J, Jiang BH, Ikeyama Y, Dida FA, Yamamoto H, Imanaka-Yoshida K, Shimpo H, Mizoguchi A, Maruyama K, Komada Y. A nuclear factor-kappaB inhibitor pyrrolidine dithiocarbamate ameliorates pulmonary hypertension in rats. Chest. 2007; 132:1265-74 Dai M, Xiao R, Cai L, Ge T, Zhu L, Hu Q. HMGB1 is mechanistically essential in the development of experimental pulmonary hypertension. Am J Physiol Cell Physiol. 2019;316:C175-85. Qin Y, Zhou A, Ben X, et al. All-trans retinoic acid in pulmonary vascular structural remodeling in rats with pulmonary hypertension induced by monocrotaline. Chinese Med J. 2001; 114: 462-5. Rosenberg HC, Rabinovitch M. Endothelial injury and vascular reactivity in monocrotaline pulmonary hypertension. Am J Physiol. 1988; 255:H1484-91 Voelkel NF, Tuder RM, Bridges J, Arend WP. Interleukin-1 receptor antagonist treatment reduces pulmonary hypertension generated in rats by monocrotaline. Am J Respir Cell Mol Biol. 1994;11:664-75. Kimura H, Kasahara Y, Kurosu K, Sugito K, Takiguchi Y, Terai M, Mikata A, Natsume M, Mukaida N, Matsushima K, Kuriyama T. Alleviation of monocrotaline-induced pulmonary hypertension by antibodies to monocyte chemotactic and activating factor/monocyte chemoattractant protein-1. Lab Invest. 1998;78:571-81. Le Cras TD, Xue C, Renqasamy A, et al. Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am J Physiol 1996; 270: L164-170 Yokochi A, Itoh H, Maruyama J, Zhang E, Jiang B, Mitani Y, Hamada C, Maruyama K. Colforsin-induced vasodilation in chronic hypoxic pulmonary hypertension in rats. J Anesth. 2010;24:432-40. Otsuka Pharmaceutical Co. LTD. Standard Commondity Classification Number of Japan 873399. PLETAAL R OD Tablets 50mg・100mg, Medical attachment, HD89D2B01, 2019 Tyler RC, Muramatsu M, Abman SH, et al. Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension. Am J Physiol 1999; 276: L297-303 Kawabe-Yako R, Ii M, Masuo O, Asahara T, Itakura T. Cilostazol activates function of bone marrow-derived endothelial progenitor cell for re-endothelialization in a carotid balloon injury model. PLoS One. 2011;6:e24646. Kimura T, Hamazaki TS, Sugaya M, Fukuda S, Chan T, Tamura-Nakano M, Sato S, Okochi H. Cilostazol improves lymphatic function by inducing proliferation and stabilization of lymphatic endothelial cells. J Dermatol Sci. 2014;74:150-8. Rabinovitch M, Gamble WJ, Miettinen OS, Reid L. Age and sex influence on pulmonary hypertension of chronic hypoxia and on recovery. Am J Physiol. 1981;240:H62-72. Zhang XP, Hintze TH. cAMP signal transduction induces eNOS activation by promoting PKB phosphorylation. Am J Physiol Heart Circ Physiol. 2006;290:H2376-84. Beg AA, Finco TS, Nantermet PV, Baldwin AS Jr. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol Cell Biol. 1993;13:3301-10 Chang KC. Cilostazol inhibits HMGB1 release in LPS-activated RAW 264.7 cells and increases the survival of septic mice. Thromb Res. 2015;136:456-64. Jung WK, Lee DY, Park C, Choi YH, Choi I, Park SG, Seo SK, Lee SW, Yea SS, Ahn SC, Lee CM, Park WS, Ko JH, Choi IW. Cilostazol is anti-inflammatory in BV2 microglial cells by inactivating nuclear factor-kappaB and inhibiting mitogen-activated protein kinases. Br J Pharmacol. 2010;159:1274-85. Park WS, Jung WK, Lee DY, Moon C, Yea SS, Park SG, Seo SK, Park C, Choi YH, Kim GY, Choi JS, Choi IW. Cilostazol protects mice against endotoxin shock and attenuates LPS-induced cytokine expression in RAW 264.7 macrophages via MAPK inhibition and NF-kappaB inactivation: not involved in cAMP mechanisms. Int Immunopharmacol. 2010;10:1077-85. O'Kane CM, Elkington PT, Jones MD, Caviedes L, Tovar M, Gilman RH, Stamp G, Friedland JS. STAT3, p38 MAPK, and NF-kappaB drive unopposed monocyte-dependent fibroblast MMP-1 secretion in tuberculosis. Am J Respir Cell Mol Biol. 2010;43:465-74. Yu BC, Lee DS, Bae SM, Jung WK, Chun JH, Urm SH, Lee DY, Heo SJ, Park SG, Seo SK, Yang JW, Choi JS, Park WS, Choi IW. The effect of cilostazol on the expression of matrix metalloproteinase-1 and type I procollagen in ultraviolet-irradiated human dermal fibroblasts. Life Sci. 2013;92:282-8. Nakamura K, Hatano E, Miyagawa-Hayashino A, Okuno M, Koyama Y, Narita M, Seo S, Taura K, Uemoto S. Soluble thrombomodulin attenuates sinusoidal obstruction syndrome in rat through suppression of high mobility group box 1. Liver Int. 2014;34:1473-87. Liver Int. 2014;34:1473-87. Tables Due to technical limitations, table 1 is only available as a download in the Supplemental Files section. Supplementary Files Table.1.pdf AuthorChecklistE10only.pdf renamedf9a91.pdf Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2021 Read the published version in BMC Pulmonary Medicine → Version 1 posted Review # 2 received at journal 07 Mar, 2021 Editorial decision: Major revision 07 Mar, 2021 Review # 1 received at journal 02 Mar, 2021 Reviewer # 2 agreed at journal 09 Feb, 2021 Reviewer # 1 agreed at journal 09 Feb, 2021 Submission checks completed at journal 09 Feb, 2021 Editor invited by journal 08 Feb, 2021 Editor assigned by journal 08 Feb, 2021 Reviewers invited by journal 08 Feb, 2021 First submitted to journal 29 Jan, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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carotid arteries was performed 21 days after the MCT or saline injection; awake mean pulmonary artery pressure (mPAP) and mean artery pressure (mAP) were measured at day 22 with rats fully awake; lung and heart samples were obtained for right ventricle (RV) weight-to-left ventricle+septum (LV+S) weight ratio (RV/LV+S), morphometry of pulmonary arteries, Western blotting and PCR after the pressure measurements. \nB, MCT28 model: systolic right ventricular pressure (sRVP) under anesthesia and sampling for RV/LV+S. Western blotting and PCR were performed 28 days after the injection of MCT or saline.\nSal/CLZ-, rats injected with saline and fed rat chow without cilostazol (CLZ)\nSal/CLZ+, rats injected with saline and fed rat chow with CLZ\nMCT/CLZ-, rats injected with MCT and fed rat chow without CLZ\nMCT/CLZ+, rats injected with MCT and fed rat chow with CLZ\nMCT, monocrotaline; Sal, saline; n=, number of rats used. We could not always succeed in obtaining all these data sets or samples for each assigned rat because of technical reasons, especially in taking mPAP, so the number of rats used (n) was not always the same as the number in Figures 5, 6, 8, and 9.","description":"","filename":"Figure.1.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/f2ef8611857dc18dbe6f70da.png"},{"id":5911834,"identity":"77ecdaf6-0c10-4584-b69b-55052fe21b86","added_by":"auto","created_at":"2021-02-12 18:16:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":56341,"visible":true,"origin":"","legend":"Experimental protocol of CH model and survival study\nA, CH model: catheterization of pulmonary and carotid arteries was performed on the final day of chronic hypoxia (CH) exposure; awake mean pulmonary artery pressure (mPAP) and mean artery pressure (mAP) were measured on day 15 with rats fully awake; lung and heart samples were obtained for right ventricle weight-to-left ventricle+septum weight ratio (RV/LV+S), morphometry of pulmonary arteries, Western blotting and PCR after the pressure measurements.\nAir/CLZ-, rats exposed to ambient air and fed rat chow without CLZ\nAir/CLZ+, rats exposed to ambient air and fed rat chow with CLZ\nCH/CLZ-, rats exposed to chronic hypoxia and fed rat chow without CLZ\nCH/CLZ+, rats exposed to chronic hypoxia and fed rat chow with CLZ\nn=, number of rats used. The number of rats used (n) was not always the same as the number in Figures 5, 6, 8, and 9. See the legend of Figure 1.\nB, Survival experiment.\nMCT/CLZ-, rats injected with MCT and fed rat chow without CLZ\nMCT/CLZ+, rats injected with MCT and fed rat chow with CLZ\nMCT, monocrotaline; Sal, saline; N=, number of rats used.\n","description":"","filename":"Figure.2.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/9ac2a307a6d5e25214bc4fc8.png"},{"id":5912181,"identity":"5b5352f4-9b8f-43c6-8438-e595b0a3689c","added_by":"auto","created_at":"2021-02-12 18:19:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":64254,"visible":true,"origin":"","legend":"Body weight\nA Rats injected with monocrotaline (MCT)\nSal/CLZ-, rats injected with saline and fed rat chow without cilostazol (CLZ)\nSal/CLZ+, rats injected with saline and fed rat chow with CLZ\nMCT/CLZ-, rats injected with MCT and fed rat chow without CLZ\nMCT/CLZ+, rats injected with MCT and fed rat chow with CLZ\n(n)= number of rats, mean ± SE\n✱ p\u003c0.05, comparison between the Sal/CLZ- group and the MCT/CLZ- group\nB Rats exposed to chronic hypoxia (CH)\nAir/CLZ-, rats exposed to ambient air and fed rat chow without CLZ\nAir/CLZ+, rats exposed to ambient air and fed rat chow with CLZ\nCH/CLZ-, rats exposed to chronic hypoxia and fed rat chow without CLZ\nCH/CLZ+, rats exposed to chronic hypoxia and fed rat chow with CLZ\n(n)= number of rats, mean ± SE\n✱ p\u003c0.05, comparison between the air/CLZ- group and the CH/CLZ- group\n","description":"","filename":"Figure.3.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/89aa4392c2010279c2c5f397.png"},{"id":5912178,"identity":"f6b68131-4e98-47d8-acd5-e6a3bda724fc","added_by":"auto","created_at":"2021-02-12 18:19:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":44716,"visible":true,"origin":"","legend":"Dosage of CLZ calculated by food intake\nA Rats injected with monocrotaline (MCT)\nEach plot is the average dosage per kg per day (from day 0 to day 5) or per 2 days (from day 7 to day 21) for a rat. For example, the plot of day 0 is the dosage a rat had taken for 24 hr from day -1 to day 0, and the plot of day 21 is the dosage a rat had taken for 48 hr from day 19 to day 21.\nSal/CLZ+, rats injected with saline and fed rat chow with CLZ\nMCT/CLZ+, rats injected with MCT and fed rat chow with CLZ\nB Rats exposed to chronic hypoxia (CH)\nEach plot is the average dosage per kg per day. For example, the plot of day 0 is the dosage a rat had taken for 24 hr from day -1 to day 0, and the plot of day 14 is the dosage a rat had taken for 24 hr from day 13 to day 14.\nAir/CLZ+, rats exposed to ambient air and fed rat chow with CLZ\nCH/CLZ+, rats exposed to chronic hypoxia and fed rat chow with CLZ\n","description":"","filename":"Figure.4.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/0926d33749bbcc035fa6fa06.png"},{"id":5912177,"identity":"55875773-551e-46a6-bb21-e1d50f96791c","added_by":"auto","created_at":"2021-02-12 18:19:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":61935,"visible":true,"origin":"","legend":"Mean pulmonary artery pressure (mPAP), ratio of mPAP to mean artery pressure (mPAP/mAP), and right ventricle (RV) weight-to-left ventricle+septum (LV+S) weight ratio (RV/LV+S)\nLeft column (MCT21) is in rats in MCT21 model.\nRight column (CH) shows rats in the CH model (rats exposed to chronic hypoxia for 14 days)\nCLZ-, fed rat chow without CLZ\nCLZ+, fed rat chow with CLZ\nSal, injected with saline\nAir, exposed to ambient air for 14 days\nMCT, monocrotaline; CH, chronic hypoxia\n(n)= number of rats, mean ± SE The number in parentheses is the number of rats in which mPAP was successfully measured.\n","description":"","filename":"Figure.5.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/c6de0b9893bc85ae2a3886ce.png"},{"id":5912714,"identity":"38d01924-2ae1-4ca9-9e2e-cdef967be0ca","added_by":"auto","created_at":"2021-02-12 18:22:20","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":39278,"visible":true,"origin":"","legend":"Systolic right ventricular pressure (sRVP) and right ventricle (RV) weight-to-left ventricle+septum (LV+S) weight ratio (RV/LV+S)\nsRVP under anesthesia and RV/LV+S were measured in rats 28 days after the injection of monocrotaline (MCT28).\nSal, injected with saline; MCT, injected with monocrotaline (MCT); CLZ-, fed rat chow without CLZ; CLZ+, fed rat chow with CLZ.\n(n) = number of rats, mean ± SE\n","description":"","filename":"Figure.6.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/4c820944ee0f607014aab6a3.png"},{"id":5911848,"identity":"e76ce4ec-584b-4b5d-bd16-17e5c1f262cb","added_by":"auto","created_at":"2021-02-12 18:16:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":32231,"visible":true,"origin":"","legend":"Kaplan-Meier survival curve of rats with and without CLZ treatment after MCT injection\nThirty rats with CLZ (MCT/CLZ+) (red line) and 30 rats without CLZ (MCT/CLZ-) (blue line) were used.\nMCT/CLZ-, rats injected with MCT and fed rat chow without CLZ\nMCT/CLZ+, rats injected with MCT and fed rat chow with CLZ\nP\u003c0.05. compared to rats without CLZ\n","description":"","filename":"Figure.7.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/f9559b6569b52ba5ea449e76.png"},{"id":5911843,"identity":"426589c5-998a-4fd6-980a-8085faf3aaa9","added_by":"auto","created_at":"2021-02-12 18:16:17","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":52150,"visible":true,"origin":"","legend":"%Muscularization and %MWT in rats injected with monocrotaline (MCT).\nThe data were obtained in MCT21 cells (rats fed with and without CLZ for 21 days after the injection of MCT).\n%Muscularization, the percentages of muscularized arteries in peripheral pulmonary arteries with an external diameter between 15 and 50 µm (A) and those between 51 and 100 µm (B).\n%MWT, %Medial wall thickness, is the ratio of the wall thickness of the media (distance between external and internal elastic laminae) to the external diameter in muscular arteries with an external diameter between 51 and 100 µm (C) and those between 101 and 200 µm (D).\nSal, injected with saline; MCT, injected with monocrotaline (MCT); CLZ-, fed rat chow without CLZ; CLZ+, fed rat chow with CLZ.\n(n)= number of rats, mean ± SE\n","description":"","filename":"Figure.8.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/05b55b8145e070adf8afd9a1.png"},{"id":5911846,"identity":"d13ee933-7629-4db7-be15-47ed69109708","added_by":"auto","created_at":"2021-02-12 18:16:17","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":46492,"visible":true,"origin":"","legend":"%Muscularization and %MWT in rats exposed to chronic hypoxia\nThe data were obtained in rats exposed to chronic hypoxia (CH) for 14 days.\nAir, exposed to ambient air; CH, rats exposed to chronic hypoxia; CLZ-, fed rat chow without CLZ; CLZ+, fed rat chow with CLZ.\nSee Figure 6 for other abbreviations\n","description":"","filename":"Figure.9.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/a961b72f8d12945945932dec.png"},{"id":5912184,"identity":"8540cb58-c50a-421a-94fc-080bd1a8c5db","added_by":"auto","created_at":"2021-02-12 18:19:17","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":280812,"visible":true,"origin":"","legend":"Endothelial nitric oxide synthase (eNOS) and phosphorylated eNOS protein levels and eNOS mRNA\nLeft column (MCT21) is in rats in MCT21 model\nMiddle column (MCT28) is in rats in MCT28 model\nRight column (CH) is in rats in CH model\nRepresentative images of Western blot showed eNOS and peNOS protein. (A, B, C, D, E, F)\nSal, injected with saline; MCT, injected with monocrotaline (MCT). Air, exposed to ambient air; CH, rats exposed to chronic hypoxia; CLZ-, fed rat chow without CLZ; CLZ+, fed rat chow with CLZ.\nThe average intensity of Sal/CLZ- in MCT21 and MCT28 cells and Air/CLZ- in CH cells was taken as 100%. Each sample intensity as a percentage of the average value was calculated (relative intensity). (n)= number of rats, mean ± SE\n\n","description":"","filename":"Figure.10.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/20b5605f43f5e626ad933dac.png"},{"id":5911849,"identity":"2ea28b20-35d0-4804-a0ee-d5a460143716","added_by":"auto","created_at":"2021-02-12 18:16:17","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":243668,"visible":true,"origin":"","legend":"AKT and phosphorylated AKT (pAKT) protein levels and AKT mRNA\nRepresentative images of Western blot showed AKT and pAKT protein. (A, B, C, D, E, F)\nSal, injected with saline; MCT, injected with monocrotaline (MCT). Air, exposed to ambient air; CH, rats exposed to chronic hypoxia; CLZ-, fed rat chow without CLZ; CLZ+, fed rat chow with CLZ.\n(n)= number of rats, mean ± SE\nSee Figure 10 for abbreviations.\n","description":"","filename":"Figure.11.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/cd2a38b219ba5fdfece15d01.png"},{"id":5911841,"identity":"aca64589-43be-4512-98fb-fe9deffebce8","added_by":"auto","created_at":"2021-02-12 18:16:17","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":235875,"visible":true,"origin":"","legend":"Western blot analysis of IκB and HMGB-1\nRepresentative images of Western blot showed IκB and HMGB-1. (A, B, C, D, E, F)\nSal, injected with saline; MCT, injected with monocrotaline (MCT). Air, exposed to ambient air; CH, rats exposed to chronic hypoxia; CLZ-, fed rat chow without CLZ; CLZ+, fed rat chow with CLZ.\nHMGB1, High-mobility group box-1.\n(n)= number of rats, mean ± SE\nSee Figure 10 for abbreviations\n\n","description":"","filename":"Figure.12.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/307d9d793d1d8a789d53f0dd.png"},{"id":5911844,"identity":"d483e8a1-f915-4c38-a455-f3b7b28561b1","added_by":"auto","created_at":"2021-02-12 18:16:17","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":32732,"visible":true,"origin":"","legend":"MCP-1 mRNA\nSal, injected with saline; MCT, injected with monocrotaline (MCT). Air, exposed to ambient air; CH, rats exposed to chronic hypoxia; CLZ-, fed rat chow without CLZ; CLZ+, fed rat chow with CLZ.\nMCP-1, Monocyte chemotactic protein-1.\n(n)= number of rats, mean ± SE\nSee Figure 10 for abbreviations\n","description":"","filename":"Figure.13.png","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/8f603680e592412f238a9acc.png"},{"id":15739396,"identity":"8ec85e2e-99be-43db-a082-e4162411d126","added_by":"auto","created_at":"2021-11-20 11:48:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2482072,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/7e216929-334e-49c1-a8eb-7d419929a1aa.pdf"},{"id":5912709,"identity":"a82bbf71-0b46-496b-9d96-6f5675944138","added_by":"auto","created_at":"2021-02-12 18:22:17","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":56117,"visible":true,"origin":"","legend":"","description":"","filename":"Table.1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/60b7d09a006a33161ce05d5a.pdf"},{"id":5912179,"identity":"a38e4e86-de8b-4b18-bb4e-f0bac294c10b","added_by":"auto","created_at":"2021-02-12 18:19:17","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":142852,"visible":true,"origin":"","legend":"","description":"","filename":"AuthorChecklistE10only.pdf","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/7489448fe5902d30eb4b5e3f.pdf"},{"id":5912707,"identity":"0cd11f5c-93b3-43de-a13c-5f2491226fe9","added_by":"auto","created_at":"2021-02-12 18:22:17","extension":"pdf","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":292576,"visible":true,"origin":"","legend":"","description":"","filename":"renamedf9a91.pdf","url":"https://assets-eu.researchsquare.com/files/rs-228016/v1/7b1ef436f4fc0ac83d65a167.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eModel Difference in the Effect of Cilostazol on the Development of Experimental Pulmonary Hypertension in Rats\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003ePulmonary hypertension (PH) is characterized by an increase in pulmonary artery pressure (PAP), right ventricular hypertrophy (RVH), and functional and/or structural vascular changes [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Possible causes of PH include pulmonary vasoconstriction, diffuse micro-thromboembolism, and pulmonary vascular remodeling [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In all conditions causing PH in humans [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and experimental models [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], vascular changes include new muscularization of normally nonmuscular peripheral pulmonary arteries and medial hypertrophy of muscular arteries. PH may be encountered in the intensive care unit in patients with acute respiratory distress syndrome (ARDS) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], congenital heart disease with left-to-right shunt [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], mitral valve disease [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], and interstitial pulmonary fibrosis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], as well as after cardiothoracic surgery [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In all conditions causing PH, including ARDS in patients [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] and experimental rat models such as monocrotaline (MCT)-induced PH [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and chronic hypoxia-induced PH [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], vascular remodeling involves new muscularization of normally nonmuscular peripheral pulmonary arteries and medial hypertrophy of muscular arteries. A total of 7.46% of critically ill patients admitted to the intensive care unit met the ARDS criteria. The prevalence of PH in these ARDS patients was as high as 46.6% [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Nitric oxide (NO) is a vasodilator and suppressor of smooth muscle cell proliferation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], and the bioavailability of NO is reduced in patients with PH and in experimental PH models [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. We and others have shown that modulation to increase NO production ameliorates the development of PH and vascular remodeling [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCilostazol (CLZ) is a selective phosphodiesterase-3 inhibitor that increases intracellular cyclic AMP, which inhibits platelet aggregation and induces peripheral vasodilation. The antiplatelet agent CLZ is indicated in intermittent claudication in peripheral arterial disease [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], thrombotic complications of coronary angioplasty [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e] and secondary stroke prevention [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Through cAMP-dependent and cAMP-independent mechanisms, CLZ also causes phosphorylation of eNOS, which increases NO production in the aortas of diabetic rats [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], human aortic endothelial cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], and rat cultured smooth muscle cells [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. CLZ also has anti-inflammatory effects [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Through activating the NO synthase-NO pathway or preventing inflammatory responses, CLZ might prevent the development of PH, as suggested in an earlier study [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] in which PH was not fatal. Since the pathogenesis and severity of PH are heterogeneous [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e], we determined the effect of CLZ on the development of two established experimental models of PH: MCT-induced PH rats [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] and chronic hypoxia (CH)-induced PH rats [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The MCT-induced PH rats in this study were used as experimental fatal PH.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003eThe 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 [56] 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\u0026ndash;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.\u003c/p\u003e\n\u003ch2\u003eAnimal groups\u003c/h2\u003e\n\u003ch2\u003e\u003cem\u003eMCT21 model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eSeven-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.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCH model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eSeven-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].\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eMCT28 model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eIn 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).\u003c/p\u003e\n\u003ch2\u003emPAP in MCT21 and CH, and sRVP in MCT28\u003c/h2\u003e\n\u003cp\u003eAt 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).\u003c/p\u003e\n\u003cp\u003eIn 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).\u003c/p\u003e\n\u003ch2\u003ePreparation of lung tissue for morphometric analysis and lung sampling for protein and mRNA assays\u003c/h2\u003e\n\u003cp\u003eAfter 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.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eMorphometric analysis of pulmonary arteries\u003c/em\u003e\u003c/strong\u003e\u003cbr /\u003e 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 \u0026micro;m and those between 51 and 100 \u0026micro;m were calculated.\u0026nbsp;For muscular arteries between 51 and 100 \u0026micro;m in\u0026nbsp;diameter and those between 101 and 200 \u0026micro;m 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].\u003c/p\u003e\n\u003ch2\u003eWestern blotting for eNOS, peNOS, AKT, I\u0026kappa;B, and HMGB-1.\u003c/h2\u003e\n\u003cp\u003eFor 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-P\u003csup\u003eR\u003c/sup\u003e, 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\u0026deg;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), I\u0026kappa;B-\u0026alpha; (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 \u0026beta;-actin, and the relative fold change compared to the control group (100%) was calculated.\u003c/p\u003e\n\u003ch2\u003ecDNA preparation and PCR\u003c/h2\u003e\n\u003cp\u003eeNOS, 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 \u0026beta;-actin mRNA.\u003c/p\u003e\n\u003ch2\u003eSurvival experiment\u003c/h2\u003e\n\u003cp\u003eSixty 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.\u003c/p\u003e\n\u003ch2\u003eData analysis\u003c/h2\u003e\n\u003cp\u003eValues are expressed as the means \u0026plusmn; SE. When more than two means were compared, one-way analysis of variance was used. When significant variance was found, Fisher\u0026rsquo;s protected least significant difference test was employed to establish which groups were different. Survival was evaluated by the Breslow-Gehan-Wilcoxon test in StatView5.0\u003csup\u003eR\u003c/sup\u003e. Differences were considered significant at P\u0026lt;0.05.\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eBody weight\u003c/h2\u003e\n\u003ch2\u003e\u003cem\u003eMCT21 model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eAll rats gained body weight steadily. MCT rats had significantly lower body weights than saline control rats from day 4 to the last day of the experiment. CLZ had no effects on body weight gain in either the MCT or saline control rats (Figure 3A).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCH model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eCH rats lost weight during the first several days of hypoxia exposure but regained weight afterward. The air rats gained weight steadily. After the start of hypoxia exposure, CH rats showed significantly lower body weights than air rats. CLZ treatment had no effect on the body weight of either air or CH rats (Figure 3B).\u003c/p\u003e\n\u003ch2\u003eDosage of CLZ\u003c/h2\u003e\n\u003ch2\u003e\u003cem\u003eMCT model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eApproximate dosages (mg) of CLZ were calculated by the equation food intake (g) x 0.003 x 1000, which is the average dosage per kg per day throughout the experimental course. The dosage of CLZ was ~230 mg/kg/day in the Sal/CLZ+ group and ~200 mg/kg/day in the MCT/CLZ+ group (Figure 4A). Although the dosage decreased to 64 mg/kg/day on the day of MCT injection, the dosage was similar in the Sal/CLZ+ and MCT/CLZ+ groups from day 4 throughout the experiment.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCH model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe dosage of CLZ was ~250 mg/kg/day in the air/CLZ+ group and ~220 mg/kg/day in the CH/CLZ+ group (Figure 4B). Although the dosage decreased to 11 mg/kg/day on the first day of hypoxia exposure, the dosage was similar in the Air/CLZ+ and CH/CLZ+ groups from day 4 throughout the experiment.\u003c/p\u003e\n\u003ch2\u003emPAP and RV/LV+S, mPAP/mAP and sRVP\u003c/h2\u003e\n\u003ch2\u003e\u003cem\u003eMCT21 model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eComparing the effects of MCT administration between Sal21/CLZ- and MCT21/CLZ-, mPAP [16.56\u0026plusmn;0.73 (n=9) vs. 35.33\u0026plusmn;1.67 (n=9) mmHg (p\u0026lt;0.05) (Fig 5A)], mPAP/mAP [0.16\u0026plusmn;0.01 (n=8) vs. 0.38\u0026plusmn;0.01 (n=8) (P\u0026lt;0.05) (Fig 5B)], and RV/LV+S [0.24\u0026plusmn;0.01 (n=10) vs. 0.40\u0026plusmn;0.01 (n=18) (P\u0026lt;0.05) (Fig 5C)] were all significantly higher in MCT21/CLZ-, suggesting that MCT caused PH and RVH. Comparing the effects of CLZ treatment between MCT21/CLZ+ and MCT21/CLZ-, mPAP [25.09\u0026plusmn;1.06 (n=11) (P\u0026lt;0.05)], mPAP/mAP [0.25\u0026plusmn;0.01 (n=11)], and RV/LV+S [0.30\u0026plusmn;0.01 (n=14)] in MCT21/CLZ+ were significantly lower than those in MCT21/CLZ- (Fig. 5A, C, E), suggesting that CLZ treatment ameliorated the development of PH and RVH.\u003c/p\u003e\n\u003ch2\u003eMCT28 model\u003c/h2\u003e\n\u003cp\u003eComparing the effects of MCT administration between Sal28/CLZ- and MCT28/CLZ-, sRVP and RV/LV+S was significantly higher in MCT28/CLZ-: sRVP, 30.20\u0026plusmn;3.35 (n=3) vs. 76.63\u0026plusmn;3.20 (n=8) mmHg (p\u0026lt;0.05) (Fig 6A); RV/LV+S, 0.28\u0026plusmn;0.02 (n=5) vs. 0.63\u0026plusmn;0.04 (n=8) (P\u0026lt;0.05) (Fig 6B). Comparing the effects of CLZ treatment between MCT28/CLZ+ and MCT28/CLZ-, sRVP [35.28\u0026plusmn;1.90 (n=8) (P\u0026lt;0.05) (Fig 6A)] and RV/LV+S [0.32\u0026plusmn;0.04 (n=9) (P\u0026lt;0.05)] in MCT28/CLZ+ were significantly lower than those in MCT28/CLZ- (Fig. 6B), suggesting again that CLZ treatment ameliorated the development of PH and RVH.\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCH model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eCH caused PH with a mPAP of 29.1\u0026plusmn;1.5 mmHg (n=10) (CH/CLZ-) compared to that in Air/CLZ- 17.5\u0026plusmn;0.5 mmHg (n=10) (p\u0026lt;0.05). There were no significant differences between CH/CLZ+ and CH/CLZ- individuals (Figure 5B). RV/LV+S was higher in CH/CLZ- 0.41\u0026plusmn;0.03 (n=13) than in Air/CLZ- 0.27\u0026plusmn;0.06 (n=10) (p\u0026lt;0.05). There were no significant differences in RV/LV+S between CH/CLZ+ and CH/CLZ- (Figure 5F). The ratio of mPAP/mAP was also higher in CH/CLZ- than Air/CLZ-, with no significant difference between CH/CLZ+ and CH/CLZ- (Figure 5D).\u003c/p\u003e\n\u003ch2\u003eSurvival\u003c/h2\u003e\n\u003cp\u003eCLZ treatment significantly improved survival from 26 days after MCT injection in MCT-induced PH rats (Figure 7).\u003c/p\u003e\n\u003ch2\u003eVascular structural changes \u003c/h2\u003e\n\u003ch2\u003e\u003cem\u003eMCT21 model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe %Muscularization was higher in those with external diameters between 15 and 50 \u0026micro;m (Figure 8A) and those between 51 and 100 \u0026micro;m (Figure 8B) in MCT21/CLZ- than in Sal21/CLZ-. MCT21/CLZ+ had significantly lower %Muscularization in both sizes of arteries than MCT21/CLZ- (Figure 8A, B). The %MWT in MCT21/CLZ- was higher than that in Sal21/CLZ- in the small muscular arteries between 51 and 100 \u0026micro;m in external diameter at the alveolar duct level, and those between 101 and 200 \u0026micro;m in external diameter were usually accompanied by terminal or respiratory bronchioles (Figure 8C, D). MCT21/CLZ+ had a significantly lower %MWT than MCT21/CLZ- (Figure 8C, D).\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eCH model\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe %Muscularization (Figure 9A, B) and %MWT (Figure 9C, D) were significantly higher in CH/CLZ- than Air/CLZ-, whereas CH/CLZ+ had no significant differences compared to CH/CLZ- (Figure 9A, B, C, D).\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eHematocrit\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eHematocrit was similar among the Sal21/CLZ-, Sal21/CLZ+, MCT21/CLZ- and MCT21/CLZ+ groups: 43.9\u0026plusmn;0.7% (n=9), 41.4\u0026plusmn;1.4% (n=9), 41.3\u0026plusmn;1.5% (n=16), and 42.4\u0026plusmn;1.5% (n=14), respectively. Hematocrit in CH/CLZ- 56.1\u0026plusmn;1.6% (n=9) was significantly higher than in Air/CLZ- 42.8\u0026plusmn;1.7% (n=9). CH/CLZ+ 56.1\u0026plusmn;1.6% (n=11) had values similar to those of CH/CLZ-.\u003c/p\u003e\n\u003ch2\u003eWestern blotting and PCR\u003c/h2\u003e\n\u003ch2\u003e\u003cem\u003eeNOS and peNOS\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eMCT had no effect on eNOS protein expression (Figure 10A, B), whereas CH upregulated eNOS protein expression (Figure 10C). MCT also had no effect on eNOS mRNA levels (Figure 10G, H), whereas chronic hypoxia increased eNOS mRNA expression (Figure 10I). MCT decreased peNOS protein expression (Figure 10D, E), whereas CH did not (Figure 10F). CLZ increased eNOS mRNA levels in the MCT21 group (Figure 10G) and decreased peNOS expression in the CH group (Figure 10F).\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eAKT and pAKT\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eMCT significantly downregulated AKT protein expression in the MCT21 model (Figure 11A) and pAKT protein expression in the MCT28 model (Figure 11E). CLZ significantly attenuated this decrease in pAKT protein expression in the MCT28 model (Figure 11E) and increased AKT mRNA expression in the MCT28 model (Figure 11H). In the CH model, AKT mRNA, AKT protein and pAKT expression were unchanged, and CLZ had no effect (Figure 11C, F, I).\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eI\u0026kappa;B and HMGB-1\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eMCT downregulated I\u0026kappa;B (Figure 12A, B) and HMGB-1 (Figure 12D, E) protein expression in both the MCT21 and MCT28 models (Figure 12A, B, D, E). CLZ significantly attenuated the downregulation of both I\u0026kappa;B (Figure 12B) and HMGB-1 (Figure 12E) protein expression in the MCT28 model. In the CH model, I\u0026kappa;B and HMGB-1 protein expression was unchanged, and CLZ had no effect (Figure 12C, F).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003e\u003cem\u003eMCP-1\u003c/em\u003e\u003c/h2\u003e\n\u003cp\u003eThe mRNA expression of MCP-1 was higher in MCT21/CLZ- than in Sal21/CLZ-. There was no significant difference between MCT21/CLZ- and MCT21/CLZ+ (Figure 13A). In the MCT28 and CH models, there were no differences in MCP-1 mRNA levels among the groups (Figure 13B, C).\u003c/p\u003e"},{"header":"Discussion","content":" \u003cp\u003eThe MCT and CH models were used to determine the model difference in the effect of CLZ on the development of PH. In both the MCT model and CH model, rats developed increases in mPAP and RV/LV\u0026thinsp;+\u0026thinsp;S, suggesting the successful development of PH. CLZ treatment ameliorated the development of MCT-induced PH. Hypertensive pulmonary vascular remodeling (i.e., new muscularization of peripheral pulmonary arteries and medial hypertrophy of muscular arteries) in the MCT model was ameliorated by CLZ treatment. In the CH model, CLZ treatment did not ameliorate the development of PH or hypertensive vascular remodeling.\u003c/p\u003e \u003cp\u003eHigh hematocrit contributes to high PAP in the CH model in addition to hypertensive pulmonary vascular remodeling [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], where the decrease in hematocrit is expected to reduce PAP. Since the hematocrit in both the CH and MCT models was not changed by CLZ, attenuation of the mPAP by CLZ treatment was not due to the decrease in hematocrit. Medial hypertrophy of the muscular artery indicates the hypertrophy and hyperplasia of vascular smooth muscle cells, whereas new muscularization of normally nonmuscular arteries indicates the differentiation of pericytes to mature smooth muscle cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Previous studies showed that increasing endogenous NO could ameliorate these structural changes [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Earlier studies revealed that eNOS mRNA is increased in the lungs of CH-induced PH rats [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], consistent with the present results. Since CLZ increases NOS expression in cultured endothelium [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], we expected to observe the upregulation of NOS due to CLZ in CH lung tissue. CLZ might have no effect on NOS synthesis, at least in CH whole lung tissue, since eNOS protein levels in the lung tissue were similar between CH-induced PH rats with and without CLZ. This suggests that combined CH and CLZ treatment did not further enhance eNOS expression compared with CH-induced NOS upregulation. Although eNOS mRNA expression was increased by CLZ in the MCT21 model, which is consistent with an earlier study [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], CLZ had no effect on the protein expression of eNOS and peNOS in either model. Translation of eNOS mRNA might be impaired in the MCT model; therefore, it is difficult to explain the preventable effect of CLZ in the MCT model by increased NO production.\u003c/p\u003e \u003cp\u003eMCT-induced PH rats [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e] have been used to investigate pulmonary vascular remodeling in inflammatory-related PH, including ARDS [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. CH-induced PH [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e] is a type of PH due to hypoxia that includes patients residing at high altitude and patients with chronic obstructive pulmonary disease. Endothelial injury precedes the increase in PAP in MCT-induced PH [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e], whereas the increase in PAP precedes the development of vascular changes in CH-induced PH [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A previous study showed that chronic NO inhalation prevented the development of PH and pulmonary vascular remodeling in CH-induced PH [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] but not in MCT-induced PH [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. NO inhalation causes selective pulmonary vasodilation. Thus, the different effects of inhaled NO between PH models suggest that reversing vasoconstriction is effective in preventing the development of PH in some forms in which vasoconstriction is the initial insult. Since CLZ could not prevent the development of CH-induced PH, we speculate that CLZ has a less potent pulmonary vasodilating effect. Endothelial injury is the initial insult in MCT-induced PH [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. CLZ has been reported to promote endothelial regeneration in injured carotid arteries [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] and endothelial proliferation in lymphatics [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are several limitations in this study. First, the present results were in adult male rats, and we must be cautious in discussing neonatal and juvenile rats and/or infant and pediatric human patients, since age and sex influence pulmonary hypertension in chronic hypoxia [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], and animal models do not completely recapture human disease [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Second, the dosage of CLZ was higher in the present study than in the previous study [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. We used this concentration because chow including 0.3% CLZ was used in spontaneously hypertensive rats [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The rats in the previous study [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] did not have a lethal condition compared with the rats in the present study. Third, this study is observational and not mechanistic. CLZ enhances Akt phosphorylation in human aortic endothelial cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and in neuroblastoma cells [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In human aortic endothelial cells [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and canine coronary blood vessels [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], PI3/AKT-dependent NO production has been reported. Since attenuation of the decrease in pAKT, an active form of AKT, was detected in the MCT model with no changes in the CH model, this effect of CLZ might partly explain the model difference in the effect of CLZ. Although one of the targets of pAKT is eNOS [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e], this is not the case in the present study because we could not detect upregulation of peNOS expression.\u003c/p\u003e \u003cp\u003eAn earlier study in our laboratory showed that MCT treatment significantly reduced the protein levels of IκBα in lung tissues, which was restored by pyrrolidine dithiocarbamate (PDTC), an NFκB inhibitor [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. NFκB is a transcription factor that regulates the transcription of genes involved in inflammatory responses. The decrease in the protein levels of IκBα has been reported to be associated with the upregulation of NFκB activity [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. IκBα was unchanged in CH-induced PH rats but decreased in MCT-induced PH rats in the current study, consistent with our earlier study [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e], suggesting that inflammatory components play a greater role in the etiology of MCT than in the chronic hypoxia model. Increased mRNA expression of lung MCP-1 in the present study also supports the inflammatory component of the MCT model. The mechanism and role of MCP-1 in the inflammatory response are chemotactic and activating effects on monocytes/macrophages [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. In the MCT model, we and others have shown macrophage infiltration into the alveolar wall by 14 days after MCT injection [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. The plasma and bronchoalveolar lavage fluid (BALF) MCP-1 levels increased transiently and then returned to normal levels [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. We also showed that lung MCP-1 mRNA was elevated in MCT21 rats in the present study and showed increases in BALF MCP-1 and TNFα levels measured by ELISA in a previous study [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], suggesting the presence of inflammation in MCT rats. The anti-inflammatory effect of CLZ [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] might explain the reversal of decreased levels of IκBα in the lungs of MCT-injected rats. CLZ has been reported to inactivate NFκB [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Moreover, retinoic acid prevented the development of MCT-induced PH with the inhibition of MMP-1 [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. NFκB induces the promotion of MMP-1 [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e], and CLZ was reported to prevent MMP-1 in a cell study [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHMGB-1 is normally present as a nuclear protein and is passively released from damaged cells [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe lower expression of HMGB-1 in the lung tissue of MCT-injected rats might be due to the increased release of HMGB-1 protein into the circulation, which might reflect damage to the cells. A recent study showed that the increase in serum HMGB-1 is associated with the concurrent decrease in tissue HMGB-1 protein expression [\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. CLZ restored HMGB-1 protein expression to the control level 28 days after MCT injection, which might show an ability of CLZ to ameliorate cell damage and improve survival. Furthermore, CLZ inhibits HMGB-1 release in lipopolysaccharide-activated HMGB-1 release and increases the survival of endotoxemic mice [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn summary, the administration of CLZ prevented the development of PH in MCT-induced PH, although CH-induced PH development was not prevented by CLZ. The inhibitory effect of CLZ on the development of PH might depend on the etiology of PH, in which alterations in lung AKT, pAKT, and IκB might partly be related.\u003c/p\u003e "},{"header":"Abbreviations","content":" \u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epulmonary hypertension\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLZ\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecilostazol\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMCT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emonocrotaline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003echronic (hypobaric) hypoxia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRV/LV\u0026thinsp;+\u0026thinsp;S\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eratio of cardiac right ventricle/(left ventricle to septum)\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emPAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emean pulmonary artery pressure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003emAP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emean artery pressure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003esRVP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esystolic right ventricular pressure\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epolymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e%Muscularization\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epercentages of muscularized arteries\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e%MWT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003epercent medial wall thickness\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eeNOS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eendothelial nitric oxide synthase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAKT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eserine-threonine protein kinase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMCP-1\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003emonocyte chemotactic protein-1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e "},{"header":"Declarations","content":"\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eThe Animal Care and Use Committee of Mie University School of Medicine approved the research protocol (No. 20-34 and 20-35).\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was financially supported by grants in aid for scientific research from the Japanese Ministry of Education, Science and Culture. (Grants-in-Aid 17K11075).\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eTI and EZ contributed equally to this work, collecting and analyzing the data, and drafting the manuscript. AO, JK, JM, MK, and AO made substantial contributions to data acquisition and reviewing the manuscript. HS, AY, and YM contributed to the study design, statistical analysis, interpretation of data, and final approval of the manuscript. KM conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eWe have no financial relationships to disclose.\u003c/p\u003e\n\u003ch2\u003eAcknowledgment\u003c/h2\u003e\n\u003cp\u003eWe would like to thank Otsuka Pharmaceutical Co. for the gift of rat chow.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eArcher SL, Weir EK, Wilkins MR. Basic science of pulmonary arterial hypertension for clinicians: new concepts and experimental therapies. 2010; 121:2045-66\u003c/li\u003e\n\u003cli\u003eRabinovitch M. Molecular pathogenesis of pulmonary arterial hypertension. J Clin Invest. 2008; 118:2372-9.\u003c/li\u003e\n\u003cli\u003eRabinovitch M, Keane JF, Norwood WI, Castaneda AR, Reid L. Vascular structure in lung tissue obtained at biopsy correlated with pulmonary hemodynamic findings after repair of congenital heart defects. 1984; 69:655-67\u003c/li\u003e\n\u003cli\u003eSnow RL, Davies P, Pontoppidan H, Zapol WM, Reid L. Pulmonary vascular remodeling in adult respiratory distress syndrome. Am Rev Respir Dis. 1982;126:887-92\u003c/li\u003e\n\u003cli\u003eTomashefski JF Jr, Davies P, Boggis C, Greene R, Zapol WM, Reid LM. The pulmonary vascular lesions of the adult respiratory distress syndrome. Am J Pathol. 1983; 112:112-26.\u003c/li\u003e\n\u003cli\u003eMitani Y, Maruyama K, Sakurai M. Prolonged administration of L-arginine ameliorates chronic pulmonary hypertension and pulmonary vascular remodeling in rats. Circulation.1997;96:689-97.\u003c/li\u003e\n\u003cli\u003eKouyoumdjian C, Adnot S, Levame M, Eddahibi S, Bousbaa H, Raffestin B. Continuous inhalation of nitric oxide protects against development of pulmonary hypertension in chronically hypoxic rats. J Clin Invest. 1994; 94:578-84.\u003c/li\u003e\n\u003cli\u003eZhang E, Jiang B, Yokochi A, Maruyama J, Mitani Y, Ma N, Maruyama K.Effect of all-trans-retinoic acid on the development of chronic hypoxia-induced pulmonary hypertension. Circ J. 2010; 74:1696-703\u003c/li\u003e\n\u003cli\u003eZhang E, Maruyama J, Yokochi A, Mitani Y, Sawada H, Nishikawa M, Ma N, Maruyama K. Sarpogrelate hydrochloride, a serotonin 5HT2A receptor antagonist, ameliorates the development of chronic hypoxic pulmonary hypertension in rats. J Anesth. 2015; 29:715-23.\u003c/li\u003e\n\u003cli\u003eRabinovitch M, Gamble W, Nadas AS, Miettinen OS, Reid L. Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features. Am J Physiol. 1979;236:H818-27\u003c/li\u003e\n\u003cli\u003eMaruyama K, Ye CL, Woo M, Venkatacharya H, Lines LD, Silver MM, Rabinovitch M. Chronic hypoxic pulmonary hypertension in rats and increased elastolytic activity. Am J Physiol. 1991;261:H1716-26.\u003c/li\u003e\n\u003cli\u003eMaruyama J, Maruyama K, Mitani Y, Kitabatake M, Yamauchi T, Miyasaka K.\u0026nbsp;Continuous low-dose NO inhalation does not prevent monocrotaline-induced pulmonary hypertension in rats. Am J Physiol. 1997;272:H517-24.\u003c/li\u003e\n\u003cli\u003eIlkiw R, Todorovich-Hunter L, Maruyama K, J Shin, and M Rabinovitch. SC-39026, a serine elastase inhibitor, prevents muscularization of peripheral arteries, suggesting a mechanism of monocrotaline-induced pulmonary hypertension in rats. Circ Res 1989; 64: 814-25\u003c/li\u003e\n\u003cli\u003eYamada Y, Maruyama J, Zhang E, Okada A, Yokochi A, Sawada H, Mitani Y, Hayashi T, Suzuki K, Maruyama K. Effect of thrombomodulin on the development of monocrotaline-induced pulmonary hypertension. J Anesth. 2014;28:26-33.\u003c/li\u003e\n\u003cli\u003eMeyrick B, Gamble W, Reid L. Development of Crotalaria pulmonary hypertension: hemodynamic and structural study. Am J Physiol. 1980;239:H692-702.\u003c/li\u003e\n\u003cli\u003eStenmark KR, Meyrick B, Galie N, Mooi WJ, McMurtry IF. Animal models of pulmonary arterial hypertension: the hope for etiological discovery and pharmacological cure. Am J Physiol Lung Cell Mol Physiol. 2009;297:L1013-32.\u003c/li\u003e\n\u003cli\u003eZapol WM, Snider MT. Pulmonary hypertension in severe acute respiratory failure. N Engl J Med. 1977; 296:476-80.\u003c/li\u003e\n\u003cli\u003eMaruyama K, Nakai Y, Takeuchi M, Mizumoto T, Chikusa H, Muneyuki M. Verapamil reduced pulmonary hypertension in adult respiratory distress syndrome. J Anesth. 1994; 8:480-1.\u003c/li\u003e\n\u003cli\u003eMaruyama K, Takeuchi M, Chikusa H, Muneyuki M. Reduction of intrapulmonary shunt by low-dose inhaled nitric oxide in a patient with late-stage respiratory distress associated with paraquat poisoning. Intensive Care Med. 1995;21:778-9.\u003c/li\u003e\n\u003cli\u003eMaruyama K, Maruyama J, Utsunomiya H, Furuhashi K, Kurobuchi M, Katayama Y, Yada I, Muneyuki M. Effect of nicardipine on pulmonary hypertension after repair of congenital heart defects in early postoperative period. J Anesth. 1993; 7:95-101.\u003c/li\u003e\n\u003cli\u003eShimpo H, Mitani Y, Tanaka J, Mizumoto T, Onoda K, Tani K, Yuasa H, Yada I, Maruyama K. Inhaled low-dose nitric oxide for postoperative care in patients with congenital heart defects. Artif Organs. 1997; 21:10-3.\u003c/li\u003e\n\u003cli\u003eBreitling S, Ravindran K, Goldenberg NM, Kuebler WM. The pathophysiology of pulmonary hypertension in left heart disease. Am J Physiol Lung Cell Mol Physiol. 2015;309:L924-41.\u003c/li\u003e\n\u003cli\u003eMaruyama K, Kobayasi H, Taguchi O, Chikusa H, Muneyuki M. Higher doses of inhaled nitric oxide might be less effective in improving oxygenation in a patient with interstitial pulmonary fibrosis. Anesth Analg. 1995; 81:210-1.\u003c/li\u003e\n\u003cli\u003eZamanian RT, Haddad F, Doyle RL, Weinacker AB. Management strategies for patients with pulmonary hypertension in the intensive care unit. Crit Care Med. 2007;35:2037-50.\u003c/li\u003e\n\u003cli\u003eGoldenberg NM, Rabinovitch M, Steinberg BE. Inflammatory Basis of Pulmonary Arterial Hypertension: Implications for Perioperative and Critical Care Medicine. Anesthesiology. 2019; 131:898-907.\u003c/li\u003e\n\u003cli\u003eHill NS, Warburton RR, Pietras L, Klinger JR. Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats. J Appl Physiol. 1997;83:1209-15.\u003c/li\u003e\n\u003cli\u003eSchermuly RT, Kreisselmeier KP, Ghofrani HA, Yilmaz H, Butrous G, Ermert L, Ermert M, Weissmann N, Rose F, Guenther A, Walmrath D, Seeger W, Grimminger F. Chronic sildenafil treatment inhibits monocrotaline-induced pulmonary hypertension in rats. Am J Respir Crit Care Med. 2004;169:39-45.\u003c/li\u003e\n\u003cli\u003eDony E, Lai YJ, Dumitrascu R, Pullamsetti SS, Savai R, Ghofrani HA, Weissmann N, Schudt C, Flockerzi D, Seeger W, Grimminger F, Schermuly RT. Partial reversal of experimental pulmonary hypertension by phosphodiesterase-3/4 inhibition. Eur Respir J. 2008;31:599-610.\u003c/li\u003e\n\u003cli\u003eSchermuly RT, Kreisselmeier KP, Ghofrani HA, Samidurai A, Pullamsetti S, Weissmann N, Schudt C, Ermert L, Seeger W, Grimminger F. Antiremodeling effects of iloprost and the dual-selective phosphodiesterase 3/4 inhibitor tolafentrine in chronic experimental pulmonary hypertension. Circ Res. 2004;94:1101-8.\u003c/li\u003e\n\u003cli\u003eEddahibi S, Raffestin B, Clozel M, Levame M, Adnot S. Protection from pulmonary hypertension with an orally active endothelin receptor antagonist in hypoxic rats. Am J Physiol. 1995;268:H828-35.\u003c/li\u003e\n\u003cli\u003eZhao L, Mason NA, Morrell NW, Kojonazarov B, Sadykov A, Maripov A, Mirrakhimov MM, Aldashev A, Wilkins MR. Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation. 2001; 104:424-8.\u003c/li\u003e\n\u003cli\u003e\u0026Ntilde;amendys-Silva S.A., Santos-Mart\u0026iacute;nez L.E., Pulido T. Pulmonary hypertension due to acute respiratory distress syndrome. Braz J Med Biol Res. 2014; 47: 904\u0026ndash;910.\u003c/li\u003e\n\u003cli\u003eKlinger JR, Kadowitz PJ．The Nitric Oxide Pathway in Pulmonary Vascular Disease. Am J Cardiol. 2017; 120: S71-9.\u003c/li\u003e\n\u003cli\u003eMaruyama J, Jiang BH, Maruyama K, Takata M, Miyasaka K. Prolonged nitric oxide inhalation during recovery from chronic hypoxia does not decrease nitric oxide-dependent relaxation in pulmonary arteries. 2004;126:1919-25.\u003c/li\u003e\n\u003cli\u003eMaruyama J, Maruyama K. Impaired nitric oxide-dependent responses and their recovery in hypertensive pulmonary arteries of rats. Am J Physiol. 1994;266:H2476-88.\u003c/li\u003e\n\u003cli\u003eZhao YD, Courtman DW, Deng Y, Kugathasan L, Zhang Q, Stewart DJ. Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease. Circ Res. 2005; 96:442-50.\u003c/li\u003e\n\u003cli\u003eHiatt WR. The US experience with cilostazol in treating intermittent claudication. Suppl. 2005; 6:21-31.\u003c/li\u003e\n\u003cli\u003eTsuchikane E, Fukuhara A, Kobayashi T, Kirino M, Yamasaki K, Kobayashi T, Izumi M, Otsuji S, Tateyama H, Sakurai M, Awata N. Impact of cilostazol on restenosis after percutaneous coronary balloon angioplasty. 1999; 100:21-6\u003c/li\u003e\n\u003cli\u003eNoma K, Higashi Y. Cilostazol for treatment of cerebral infarction. Expert Opin Pharmacother. 2018; 19:1719-26.\u003c/li\u003e\n\u003cli\u003eSuzuki K, Uchida K, Nakanishi N, Hattori Y. Cilostazol activates AMP-activated protein kinase and restores endothelial function in diabetes. Am J Hypertens. 2008; 21:451-7.\u003c/li\u003e\n\u003cli\u003eHashimoto A, Miyakoda G, Hirose Y, Mori T. Activation of endothelial nitric oxide synthase by cilostazol via a cAMP/protein kinase A- and phosphatidylinositol 3-kinase/Akt-dependent mechanism. Atherosclerosis. 2006; 189:350-7.\u003c/li\u003e\n\u003cli\u003eIkeda U, Ikeda M, Kano S, Kanbe T, Shimada K. Effect of cilostazol, a cAMP phosphodiesterase inhibitor, on nitric oxide production by vascular smooth muscle cells. Eur J Pharmacol. 1996; 314:197-202.\u003c/li\u003e\n\u003cli\u003eHassan M, Ibrahim MA, Hafez HM, Mohamed MZ, Zenhom NM, Abd Elghany HM.\u0026nbsp;Role of Nrf2/HO-1 and PI3K/Akt Genes in the Hepatoprotective Effect of Cilostazol. Curr Clin Pharmacol. 2019; 14:61-7.\u003c/li\u003e\n\u003cli\u003eAndersson U, Tracey KJ. HMGB1 is a therapeutic target for sterile inflammation and infection. Annu Rev Immunol. 2011; 29:139-62.\u003c/li\u003e\n\u003cli\u003eSakamoto T, Ohashi W, Tomita K, Hattori K, Matsuda N, Hattori Y. Anti-inflammatory properties of cilostazol: Its interruption of DNA binding activity of NF-\u0026kappa;B from the Toll-like receptor signaling pathways. Int Immunopharmacol. 2018;62:120-31.\u003c/li\u003e\n\u003cli\u003eChang LT, Sun CK, Sheu JJ, Chiang CH, Youssef AA, Lee FY, Wu CJ, Yip HK.\u0026nbsp;Cilostazol therapy attenuates monocrotaline-induced pulmonary arterial hypertension in rat model. Circ J. 2008; 72:825-31.\u003c/li\u003e\n\u003cli\u003eSimonneau G, Gatzoulis MA, Adatia I, Celermajer D, Denton C, Ghofrani A, Gomez Sanchez MA, Krishna Kumar R, Landzberg M, Machado RF, Olschewski H, Robbins IM, Souza R. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol. 2013; 62: D34-41.\u003c/li\u003e\n\u003cli\u003eSawada H, Mitani Y, Maruyama J, Jiang BH, Ikeyama Y, Dida FA, Yamamoto H, Imanaka-Yoshida K, Shimpo H, Mizoguchi A, Maruyama K, Komada Y. A nuclear factor-kappaB inhibitor pyrrolidine dithiocarbamate ameliorates pulmonary hypertension in rats. Chest. 2007; 132:1265-74\u003c/li\u003e\n\u003cli\u003eDai M, Xiao R, Cai L, Ge T, Zhu L, Hu Q. HMGB1 is mechanistically essential in the development of experimental pulmonary hypertension. Am J Physiol Cell Physiol. 2019;316:C175-85.\u003c/li\u003e\n\u003cli\u003eQin Y, Zhou A, Ben X, et al. All-trans retinoic acid in pulmonary vascular structural remodeling in rats with pulmonary hypertension induced by monocrotaline. Chinese Med J. 2001; 114: 462-5.\u003c/li\u003e\n\u003cli\u003eRosenberg HC, Rabinovitch M. Endothelial injury and vascular reactivity in monocrotaline pulmonary hypertension. Am J Physiol. 1988; 255:H1484-91\u003c/li\u003e\n\u003cli\u003eVoelkel NF, Tuder RM, Bridges J, Arend WP. Interleukin-1 receptor antagonist treatment reduces pulmonary hypertension generated in rats by monocrotaline. Am J Respir Cell Mol Biol. 1994;11:664-75.\u003c/li\u003e\n\u003cli\u003eKimura H, Kasahara Y, Kurosu K, Sugito K, Takiguchi Y, Terai M, Mikata A, Natsume M, Mukaida N, Matsushima K, Kuriyama T. Alleviation of monocrotaline-induced pulmonary hypertension by antibodies to monocyte chemotactic and activating factor/monocyte chemoattractant protein-1. Lab Invest. 1998;78:571-81.\u003c/li\u003e\n\u003cli\u003eLe Cras TD, Xue C, Renqasamy A, et al. Chronic hypoxia upregulates endothelial and inducible NO synthase gene and protein expression in rat lung. Am J Physiol 1996; 270: L164-170\u003c/li\u003e\n\u003cli\u003eYokochi A, Itoh H, Maruyama J, Zhang E, Jiang B, Mitani Y, Hamada C, Maruyama K. Colforsin-induced vasodilation in chronic hypoxic pulmonary hypertension in rats. J Anesth. 2010;24:432-40.\u003c/li\u003e\n\u003cli\u003eOtsuka Pharmaceutical Co. LTD. Standard Commondity Classification Number of Japan 873399. PLETAAL\u003csup\u003eR\u003c/sup\u003e OD Tablets 50mg・100mg, Medical attachment, HD89D2B01, 2019\u003c/li\u003e\n\u003cli\u003eTyler RC, Muramatsu M, Abman SH, et al. Variable expression of endothelial NO synthase in three forms of rat pulmonary hypertension. Am J Physiol 1999; 276: L297-303\u003c/li\u003e\n\u003cli\u003eKawabe-Yako R, Ii M, Masuo O, Asahara T, Itakura T. Cilostazol activates function of bone marrow-derived endothelial progenitor cell for re-endothelialization in a carotid balloon injury model. PLoS One. 2011;6:e24646.\u003c/li\u003e\n\u003cli\u003eKimura T, Hamazaki TS, Sugaya M, Fukuda S, Chan T, Tamura-Nakano M, Sato S, Okochi H. Cilostazol improves lymphatic function by inducing proliferation and stabilization of lymphatic endothelial cells. J Dermatol Sci. 2014;74:150-8.\u003c/li\u003e\n\u003cli\u003eRabinovitch M, Gamble WJ, Miettinen OS, Reid L. Age and sex influence on pulmonary hypertension of chronic hypoxia and on recovery. Am J Physiol. 1981;240:H62-72.\u003c/li\u003e\n\u003cli\u003eZhang XP, Hintze TH. cAMP signal transduction induces eNOS activation by promoting PKB phosphorylation. Am J Physiol Heart Circ Physiol. 2006;290:H2376-84.\u003c/li\u003e\n\u003cli\u003eBeg AA, Finco TS, Nantermet PV, Baldwin AS Jr. Tumor necrosis factor and interleukin-1 lead to phosphorylation and loss of I kappa B alpha: a mechanism for NF-kappa B activation. Mol Cell Biol. 1993;13:3301-10\u003c/li\u003e\n\u003cli\u003eChang KC. Cilostazol inhibits HMGB1 release in LPS-activated RAW 264.7 cells and increases the survival of septic mice. Thromb Res. 2015;136:456-64.\u003c/li\u003e\n\u003cli\u003eJung WK, Lee DY, Park C, Choi YH, Choi I, Park SG, Seo SK, Lee SW, Yea SS, Ahn SC, Lee CM, Park WS, Ko JH, Choi IW. Cilostazol is anti-inflammatory in BV2 microglial cells by inactivating nuclear factor-kappaB and inhibiting mitogen-activated protein kinases. Br J Pharmacol. 2010;159:1274-85.\u003c/li\u003e\n\u003cli\u003ePark WS, Jung WK, Lee DY, Moon C, Yea SS, Park SG, Seo SK, Park C, Choi YH, Kim GY, Choi JS, Choi IW. Cilostazol protects mice against endotoxin shock and attenuates LPS-induced cytokine expression in RAW 264.7 macrophages via MAPK inhibition and NF-kappaB inactivation: not involved in cAMP mechanisms. Int Immunopharmacol. 2010;10:1077-85.\u003c/li\u003e\n\u003cli\u003eO'Kane CM, Elkington PT, Jones MD, Caviedes L, Tovar M, Gilman RH, Stamp G, Friedland JS. STAT3, p38 MAPK, and NF-kappaB drive unopposed monocyte-dependent fibroblast MMP-1 secretion in tuberculosis. Am J Respir Cell Mol Biol. 2010;43:465-74.\u003c/li\u003e\n\u003cli\u003eYu BC, Lee DS, Bae SM, Jung WK, Chun JH, Urm SH, Lee DY, Heo SJ, Park SG, Seo SK, Yang JW, Choi JS, Park WS, Choi IW. The effect of cilostazol on the expression of matrix metalloproteinase-1 and type I procollagen in ultraviolet-irradiated human dermal fibroblasts. Life Sci. 2013;92:282-8.\u003c/li\u003e\n\u003cli\u003eNakamura K, Hatano E, Miyagawa-Hayashino A, Okuno M, Koyama Y, Narita M, Seo S, Taura K, Uemoto S. Soluble thrombomodulin attenuates sinusoidal obstruction syndrome in rat through suppression of high mobility group box 1. Liver Int. 2014;34:1473-87. Liver Int. 2014;34:1473-87.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eDue to technical limitations, table 1 is only available as a download in the Supplemental Files section.\u003c/p\u003e\n"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-pulmonary-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pulm","sideBox":"Learn more about [BMC Pulmonary Medicine](http://bmcpulmmed.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pulm/default.aspx","title":"BMC Pulmonary Medicine","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"monocrotaline, chronic hypoxia, cilostazol, pulmonary hypertension, nitric oxide","lastPublishedDoi":"10.21203/rs.3.rs-228016/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-228016/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003e\u003cem\u003eBackground:\u003c/em\u003e \u003c/strong\u003ePreventing pulmonary vascular remodeling is a key strategy for pulmonary hypertension (PH). Causes of PH include pulmonary vasoconstriction and inflammation. This study aimed to determine whether cilostazol (CLZ), a phosphodiesterase-3 inhibitor, prevents monocrotaline (MCT)- and chronic hypoxia (CH)-induced PH development in rats.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eMethods: \u003c/em\u003e\u003c/strong\u003eFifty-one male Sprague-Dawley rats were fed rat chow with (0.3% CLZ) or without CLZ for 21 days after a single injection of MCT (60 mg/kg) or saline. Forty-eight rats were fed rat chow with and without CLZ for 14 days under ambient or hypobaric (air at 380 mmHg) CH exposure. Mean PAP (mPAP), the right ventricle weight-to-left ventricle+septum weight ratio (RV/LV+S), percentages of muscularized peripheral pulmonary arteries (%Muscularization) and medial wall thickness of small muscular arteries (%MWT) were assessed.\u003c/p\u003e\u003cp\u003eProtein expression of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (peNOS), AKT, pAKT and IκB in lung tissue was measured by Western blotting. Monocyte chemotactic protein (MCP)-1 mRNA in lung tissue was also assessed.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eResults: \u003c/em\u003e\u003c/strong\u003emPAP [35.1±1.7 mmHg (MCT) (n=9) vs.16.6±0.7 (control) (n=9) (p\u0026lt;0.05); 29.1±1.5 mmHg (CH) (n=10) vs. 17.5±0.5 (control) (n=10) (p\u0026lt;0.05)], RV/LV+S [0.40±0.01 (MCT) (n=18) vs. 0.24±0.01 (control) (n=10) (p\u0026lt;0.05); 0.41±0.03 (CH) (n=13) vs. 0.27±0.06 (control) (n=10) (p\u0026lt;0.05)], and %Muscularization and %MWT were increased by MCT injection and CH exposure. CLZ significantly attenuated these changes in the MCT model [mPAP 25.1±1.1 mmHg (n=11) (p\u0026lt;0.05), RV/LV+S 0.30±0.01 (n=14) (p\u0026lt;0.05)]. In contrast, these CLZ effects were not observed in the CH model. Lung eNOS protein expression was unchanged in the MCT model and high in the CH model. Lung protein expression of AKT, phosphorylated AKT, and IκB was downregulated by MCT, which was attenuated by CLZ; the CH model did not change these proteins. Lung MCP-1 mRNA levels were increased in MCT rats but not CH rats.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003e\u003cem\u003eConclusion: \u003c/em\u003e\u003c/strong\u003eWe found model differences in the effect of CLZ on PH development. CLZ might have a preventable effect on PH development in an inflammatory PH model but not in a vascular structural change model of PH preceded by vasoconstriction. Thus, the preventive effect of CLZ on PH development might be dependent on PH etiology.\u003c/p\u003e","manuscriptTitle":"Model Difference in the Effect of Cilostazol on the Development of Experimental Pulmonary Hypertension in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-02-12 18:16:14","doi":"10.21203/rs.3.rs-228016/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-03-08T00:00:00+00:00","index":2,"fulltext":"Recommendation: Major revisions required\nForm responses:\n---\n\nComments to Author:\n---\nThis article by Ito and co-authors describes the effects of cilostazol (CLZ) on two rat models of pulmonary hypertension (PH), chronic hypoxia (CH) and monocrotaline-induced (MCT). The rationale behind this is because CLZ is a phosphodiesterase type-5 inhibitor with anti-inflammatory and antiplatelet capabilities, shown to prevent PH as per the Chang study in 2008.\n\nExperiments were done in 7-week old Sprague-Dawley rats (all male). Animals were fed with CLZ daily, for 21 days or 28 days to measure systolic right ventricular pressure (sRVP). Hypoxic rats were kept in hypobaric hypoxic conditions for 2 weeks and then returned to room air. In the MCT-28 model, they measured mPAP on awake animals. They measured mPAP and Fulton index, as well as medial wall thickness, eNOS protein expression, AKT, an iKB, MCP-1 mRNA.\n\nThe authors concluded that CLZ treatments ameliorated the development of PH by decreasing mPAP, Fulton index and hypertensive pulmonary vascular remodeling on the MCT but not in CH rats. CLZ had no effects in hematocrit, eNOS, AKT, nor in Ikb. Authors acknowledge the observational nature of this study and propose that improvements may depend on the etiology of PH. Some mechanistic digging would improve this study significantly.\n\nRegarding specific findings and some other recommendations:\n1. the authors report that all rats gained weight steadily and that CLZ had no effects on weight, which seems to be well supported by the data. It seems though, that rats randomized into the MCT21 group were heavier (from baseline to end, including the control animals). I am curious to know why this is; the authors should clarify.\n2. CLZ improved mPAP and Fulton index in MCT21 but not in CH rats. Nevertheless, the parameters still remained high, compared to controls; the authors should comment on this.\n3. CLZ helped rats to survive 4 additional days, compared to CLZ. It is suggested that the single sentence describing that finding be added to the relevant paragraph, rather than making it a separate section.\n4. The authors state that the % muscularization in MCT21 were lowered significantly in CLZ-treated rats however, there is no indication of statistical significance in the figure (8A). Also, if the data are presented as mean  SE, how come data in Fig 8C is significant as claimed in page 17 lines 17-18? Please provide p values and statistical test used.\n5. Authors acknowledge that sex and age influence PH but did not offer a reason/justification for using all males in the study.\n6. Please include graphical representation of the hematocrit data, even if not significant differences were found.\n7. I strongly recommend providing complete labeling of axes in the figures. For instance, panels to the right in Figure 5 (B, D, and F) are missing the numeric labels. This makes it very hard to the reader. Same applies to Figs. 8, 9, 10, 11, 12. 13.\n8. Figure 8: vessel muscularization in control animals is invisible, given higher numbers in the treated animals and the range of the dataset. I suggest using broken axes so that we can better appreciate the control data. Same for MCP1 data in Figure 13 form MCT28 and CH\n9. There is repetition in page 5, lines 10-14. Please revise.\n10. Rearrangements in the Discussion section may improve the flow. For example, I would move the statements of limitations (page 22, line 12 and on) to the penultimate paragraph.\n\n\n* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons after the final decision on the manuscript has been made. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **No**\n* Declaration of competing interests: **I declare that I have no competing interests**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. (default)**\n* Is the study design appropriate to answer the research question (including the use of appropriate controls), and are the conclusions supported by the evidence presented?: **Yes**\n* Are the methods sufficiently described to allow the study to be repeated?: **Yes**\n* Is the use of statistics and treatment of uncertainties appropriate?: **Yes**\n* Is the presentation of the work clear?: **Yes**\n* Are the images in this manuscript (including electrophoretic gels and blots) free from apparent manipulation?: **Yes**\n"},{"type":"decision","content":"Major revision","date":"2021-03-08T00:00:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-03-03T00:00:00+00:00","index":1,"fulltext":"Recommendation: Major revisions required\nForm responses:\n---\n\nComments to Author:\n---\nThis manuscript from Dr. Ito and colleagues studies the effect of preventive cilostazol treatment on the development of experimental pulmonary hypertension induced by monocrotaline (MCT) and chronic hypoxia (CH) in rats. The authors found that cilostazol reduced the development of MCT-induced PH, but not CH-induced PH. The authors further identified the effect of cilostazol treatment on several pathways implicated in PH pathobiology, including AKT, eNOS, NFkB/IkΒ, HMGB1 and MCP-1. They found different effects of cilostazol in MCT and CH PH. The work appears well done and new therapies for PH could help to improve patient outcome. However, there are several major and minor issues with the manuscript in its present form.\n\nMajor comments:\n- Animal models: it is unclear what the rationale was to compare preventive treatment of MCT rats for 21 and 28 days. An interventional treatment approach would represent a better translational approach, e.g., treatment start at day 21.\n- As MCT rats develop right heart failure and can also develop substantial myocardial inflammation, an assessment of right ventricular function (e.g., cardiac output) and right ventricular changes, such as apoptotic index, right ventricular fibrosis or changes in capillary density, should be also evaluated.\n- Both the MCT and the CH models lead to increased muscularization. Do the authors have an explanation why CLZ only reduced muscularization in the MCT model. Also, why is CLZ not working in the CH model?\n- The authors are using mPAP in Figure 5 and RVSP in Figure 6 - they should use the same outcome parameters in both Figures to avoid confusion of the reader.\n- The study of the molecular targets is not well rationalized and should be better explained and interpreted in the discussion. \n- The authors could tighten their story by localizing some of the key molecules that they studied using Western blots in pulmonary arteries by immunohistochemistry.\n- Were any effects of the treatment observed in MCT or CH PH models on smooth muscle cell proliferation or endothelial cell apoptosis? This could be investigated using immunohistochemistry.\n\nMinor comments:\n- Discussion page 21, line 12: \"Translation of eNOS mRNA might be impaired in the MCT model\". The data show that MCT treatment did not change lung tissue eNOS mRNA expression, but this does not mean that MCT impaired eNOS mRNA expression. Please correct the statement to reflect the actual data.\n- Page 21, line 12: please correct \"preventable effect of CLZ\" to \"preventive effect of CLZ\".* Publons Reviewer Recognition. Springer Nature can send verification of this review directly to Publons (a subsidiary of Clarivate Analytics). If you would like to take advantage of this service, please click on the “Yes” option below. Your name, email address, title of the reviewed manuscript, name of the journal, and date of your review submission (the “Review Data”) will then be transmitted to Publons after the final decision on the manuscript has been made. If you have already registered at Publons, they will notify you of the receipt of this review and update your profile as per your settings and their policy. If you are not registered with Publons, you will receive an email from them asking you to register in order for them to be able to recognize your review on your new profile page. Publons may use the Review Data to generate derivative metadata for the benefit of Publons and you as a reviewer, carefully considering the sensitivity of such information. For example, Publons may verify your record as a reviewer by updating your profile published on its webservice if you have registered for such service or help editors to identify candidate reviewers. Please find the details of processing in Publons’ privacy policy https://publons.com/about/terms: **Yes**\n* Declaration of competing interests: **I declare that I have no competing interests.**\n* Reviewer Publication Consent. I agree for my report to be made available under an Open Access Creative Commons CC-BY License (http://creativecommons.org/licenses/by/4.0) if this manuscript is accepted for publication. Any comments that I do not wish to be included in the published report have been included as confidential comments to the editor, which will not be published.: **I agree to the terms of the CC-BY 4.0 license; please do not publish my name with my report. 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