Simvastatin prevents and reverses chronic pulmonary hypertension in newborn rats via pleiotropic inhibition of RhoA signaling Simvastatin prevents and reverses chronic pulmonary hypertension in newborn rats via pleiotropic inhibition of RhoA signaling Mathew J. Wong,1,4 Crystal Kantores,1 Julijana Ivanovska,1 Amish Jain,2,3,4 and Robert P. Jankov1,2,3,4 1Physiology & Experimental Medicine Program, Hospital for Sick Children Research Institute, Toronto, Ontario, Canada; 2Heart and Stroke Richard Lewar Centre of Excellence in Cardiovascular Research, University of Toronto, Toronto, Ontario, Canada; 3Department of Paediatrics, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada; and 4Department of Physiology, Faculty of Medicine, University of Toronto, Toronto, Ontario, Canada Submitted 9 August 2016; accepted in final form 30 September 2016 Wong MJ, Kantores C, Ivanovska J, Jain A, Jankov RP. Simvastatin prevents and reverses chronic pulmonary hypertension in newborn rats via pleiotropic inhibition of RhoA signaling. Am J Physiol Lung Cell Mol Physiol 311: L985–L999, 2016. First published September 30, 2016; doi:10.1152/ajplung.00345.2016.— Chronic neonatal pulmonary hypertension (PHT) frequently results in early death. Systemically administered Rho-kinase (ROCK) inhibitors prevent and reverse chronic PHT in neonatal rats, but at the cost of severe adverse effects, including systemic hypotension and growth restriction. Simvastatin has pleiotropic inhibitory effects on iso- prenoid intermediates that may limit activity of RhoA, which signals upstream of ROCK. We therefore hypothesized that statin treatment would safely limit pulmonary vascular RhoA activity and prevent and reverse experimental chronic neonatal PHT via downstream inhibitory effects on pathological ROCK activity. Sprague-Dawley rats in nor- moxia (room air) or moderate normobaric hypoxia (13% O2) received simvastatin (2 mg·kg�1·day�1 ip) or vehicle from postnatal days 1–14 (prevention protocol) or from days 14 –21 (rescue protocol). Chronic hypoxia increased RhoA and ROCK activity in lung tissue. Simva- statin reduced lung content of the isoprenoid intermediate farnesyl pyrophosphate and decreased RhoA/ROCK signaling in the hypoxia- exposed lung. Preventive or rescue treatment of chronic hypoxia- exposed animals with simvastatin decreased pulmonary vascular re- sistance, right ventricular hypertrophy, and pulmonary arterial remod- eling. Preventive simvastatin treatment improved weight gain, did not lower systemic blood pressure, and did not cause apparent toxic effects on skeletal muscle, liver or brain. Rescue therapy with sim- vastatin improved exercise capacity. We conclude that simvastatin limits RhoA/ROCK activity in the chronic hypoxia-exposed lung, thus preventing or ameliorating hemodynamic and structural markers of chronic PHT and improving long-term outcome, without causing adverse effects. chronic lung injury; Rho-kinase; echocardiography; isoprenoid inter- mediates PREMATURE BIRTH AND ASSOCIATED chronic lung injury, known as bronchopulmonary dysplasia (BPD), is frequently associated with chronic pulmonary arterial hypertension (PHT) (3). Chronic PHT is characterized by pulmonary arterial remodel- ing, leading to increased pulmonary arterial pressure, increased pulmonary vascular resistance (PVR), and decreased arterial compliance (4, 11, 62). The normal pulmonary circulation is a low-resistance, high-compliance system suited to accommo- date the entire cardiac output. Increased thickening of pulmo- nary arteries from smooth muscle proliferation and extracellu- lar matrix deposition contributes to decreased pulmonary arte- rial compliance, increased resistance and increased right ventricle (RV) afterload (36). The RV adapts by undergoing hypertrophy that may be followed by maladaptive chamber dilatation, which precedes RV failure and eventual death (5, 6). Newborns with chronic PHT associated with severe BPD have a high mortality, with one study reporting a 2-year survival rate of 52% (3, 48). Moreover, survivors are at an increased risk for impaired neurological development (34) and recurrence of PHT later in life (58). Currently, there are no treatments of proven efficacy for chronic PHT in neonates. Previous work from our laboratory has implicated the ras- homolog gene family member A (RhoA)/Rho-kinase (ROCK) signaling pathway in the pathogenesis of experimental chronic neonatal PHT. RhoA/ROCK upregulation leads to increased pulmonary vascular tone and pulmonary arterial remodeling in chronic PHT (66). Treatment with ROCK-specific inhibitors such as fasudil and Y-27632 has been shown to prevent or reverse chronic hypoxic- or bleomycin-induced PHT in neo- natal rats (22, 39, 74, 75). In pulmonary artery smooth muscle cells (SMCs), vasoconstriction is regulated by phosphorylation of myosin light chain (MLC) through MLC kinase. Dephos- phorylation by MLC phosphatase leads to vasorelaxation (14). ROCK acts by phosphorylating the myosin phosphatase target subunit 1 (MYPT1), which inhibits MLC phosphatase activity, thus favoring a contractile state (59, 66). ROCK mediates arterial remodeling via regulation of antiapoptotic and propro- liferative signaling in SMCs and fibroblasts (23, 62, 74). RhoA/ROCK signaling also negatively regulates endothelial nitric oxide synthase (eNOS) function (28, 56). Statins [3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase inhibitors] prevent conversion of HMG-CoA to mevalonate, which is the rate-limiting step in cholesterol biosynthesis (15). Inhibition of mevalonate synthesis also de- creases the synthesis of isoprenoid intermediates (illustrated in Fig. 1A): farnesyl-pyrophosphate (FPP) and geranylgeranyl- pyrophosphate (GGPP) (21). The isoprenoids serve an essen- tial biological role through lipidation of proteins, a process also known as prenylation. Of the isoprenoid intermediates, FPP interacts with the Ras family of proteins while GGPP binds to the Rho family (38). An inactive reservoir of GDP-RhoA remains in the cytosol bound to Rho-specific guanine nucleo- tide dissociation inhibitor (RhoGDI) (18). GGPP-mediated prenylation of RhoA is required for intracellular cytosolic trafficking and subsequent activation at the membrane (38, 72). Once localized to the membrane, RhoA is activated by guanine nucleotide exchange factors catalyzing the exchange of GDP for GTP. RhoA is inactivated by GTPase-activating proteins Address for reprint requests and other correspondence: r. P. Jankov, Hospital For Sick Children, 09.9707 Peter Gilgan Centre for Research and Learning, 686 Bay St., Toronto, ON M5G 0A4, Canada (e-mail: robert.jankov@sickkids.ca). Am J Physiol Lung Cell Mol Physiol 311: L985–L999, 2016. First published September 30, 2016; doi:10.1152/ajplung.00345.2016. 1040-0605/16 Copyright © 2016 the American Physiological Societyhttp://www.ajplung.org L985 Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. mailto:robert.jankov@sickkids.ca through hydrolysis of the phosphate bond (37). GGPP- mediated prenylation of RhoA may also prevent membrane dissociation through the action of RhoGDI, further enhanc- ing RhoA activation (12). Once active, RhoA binds to the Rho-binding domain of ROCK, inducing an active confor- mational change (65). Previous work from our laboratory has confirmed the im- portance of upregulated RhoA activity and its downstream mediator ROCK in the pathogenesis of experimental chronic neonatal lung injury and chronic neonatal PHT (39, 75). Work from other investigators has demonstrated pleiotropic inhibi- tion of RhoA activity by statins (7, 33, 42). Therefore, by limiting the synthesis of GGPP and prenylation of RhoA, statins may reduce pathological RhoA and ROCK activation (72). The objective of this study was to examine the effects of simvastatin in the prevention or rescue of chronic hypoxic PHT in neonatal rats. MATERIALS AND METHODS Materials. Simvastatin was from Cayman Chemical (catalog no. 10010344, Ann Arbor, MI). Oxygen-exposure chambers and auto- mated controllers, OxyCycler model A84XOV, were from Biospherix (Parish, NY). Tris-glycine precast gels (catalog no. XP04205BOX, 15 well, 4 –20%) and PVDF membranes were from Thermo Scientific (Rockford, IL). Phosphatase and protease inhibitors were from Sigma Life Science (catalog no. P5726-5ML, St. Louis, MO) and Calbio- chem (catalog no. 539131-10VL, San Diego, CA), respectively. Ac- ids, alcohols, organic solvents, paraformaldehyde, Permount, and Superfrost/Plus microscope slides were from Fisher Scientific (Whitby, Ontario, Canada). Weigert’s resorcin-fuchsin stain was from Rowley Biochemical (catalog no. F-370-1, Danvers, MA). Luxol Fast Blue stain solutions were from Electron Microscopy Sciences (catalog no. 2668101, Hatfield, PA). ELISA kits for alanine transaminase (ALT; catalog no. ab105134), creatine kinase (CK; catalog no. ab155901), high-density lipoprotein (HDL), and low-density/very- low-density lipoprotein (LDL/VLDL) cholesterol (catalog no. ab65390) were purchased from Abcam (Toronto, Ontario, Canada). A free cholesterol assay kit (catalog no. 10007640) was from Cayman Chemical. Anti-ROCK I (catalog no. SC-5560), anti-cleaved ROCK I (catalog no. SC-52953), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; catalog no. SC-25778) antibodies and protein A/G agarose beads (catalog no. SC-2003) were from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-ROCK II (catalog no. 610624), anti-pan- MYPT1 (catalog no. 612164), and anti-eNOS (catalog on. 61098) were from BD Biosciences (San Jose, CA). Anti-phospho-threonine 850 MYPT1 (catalog no. 36-003) was from Millipore (Etobicoke, Ontario, Canada). Anti-HIF-1� (catalog no. 821503130) was from GeneTex (Irvine, CA). Anti-GTP-RhoA (catalog no. 26904) and anti-RhoA (catalog no. 21017) antibodies were from NewEast Bio- Fig. 1. Illustration of statin inhibition of cholesterol and iso- prenoid synthesis pathways. Experimental design for preven- tion and rescue protocols. A: upstream inhibition of HMG-CoA reductase enzyme limits bioavailability of diverging meval- onate products. Reduction of the isoprenoid intermediate gera- nylgeranyl pyrophosphate (GGPP) limits membrane transloca- tion and activation of RhoA. B: overview of the time course and experimental design for prevention and rescue protocols. L986 SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. sciences (King of Prussia, PA). Anti-�-smooth muscle actin antibody (catalog no. MA1-12772) was from Thermo Scientific. Avidin-biotin- peroxidase complex immunohistochemistry kits, 6-diamino-2- phenylindole aqueous mounting medium, and 3,3-diaminobenzidine staining kits were from Vector Laboratories (Burlingame, CA). Goat anti-mouse immunoglobulin (IgG)-peroxidase antibodies, and biotin- ylated goat anti-mouse IgG (catalog no. 14709) and goat anti-rabbit IgG (catalog no. 14708) were from Cell Signaling Technologies (Beverly, MA). In vivo experiments. All procedures involving animals were ap- proved by the Animal Care Committee of the Hospital for Sick Children Research Institute and conformed to the guidelines of the Canadian Council on Animal Care. Timed-pregnant Sprague-Dawley rats were from Taconic Farms (Germantown, NY). Commencing on the day after birth [postnatal day (PND) 1], litters of Sprague-Dawley rat pups (equalized sex distribution) were chronically exposed to normobaric hypoxia (13% O2) or normoxia (21% O2) until PND 14 (prevention study) or until PND 21 (rescue study). Concurrent with normoxia or hypoxia exposure, rat pups received daily intraperitoneal simvastatin (2 mg/kg) or vehicle (20% DMSO in PBS), from PND 1 to PND 14 (prevention study) or from PND 14 to PND 21 (rescue study). Injury and treatment protocols are illustrated in Fig. 1B. Simvastatin was selected as it was the most frequently studied statin in the clinical and experimental PHT literature during the conception of the study design, with a well-defined dose range in neonatal rats (21, 40, 41, 54, 63). Initial dose-response studies (0.1, 2, 5, 10, and 20 mg/kg) were conducted to determine the lowest effective dose in preventing PHT, as guided by echocardiographic estimation of PVR. The highest dose (20 mg/kg) adversely affected weight gain and the lowest dose (0.1 mg/kg) had a minimal effect on PVR (data not shown); therefore 2 mg/kg was the dose chosen for all subsequent studies. Each litter was maintained at n � 12 pups, to control for nutritional effects. At the end of each 14- or 21-day exposure period, pups either were killed by pentobarbital overdose or were exsangui- nated after anesthesia. Upon completion of the rescue protocol, a subgroup of animals was housed in room air until 10 wk of age for exercise testing. Simvastatin preparation and delivery. Simvastatin was provided as a crystallized solid and stored in aliquots as a 25 mg/ml solution in DMSO stored at �20°C. Since simvastatin is not soluble in saline, a 0.4 mg/ml solution in 20% DMSO in PBS was heated to 37°C in a temperature regulated shaker just prior to delivery to ensure solubility. Injections were given intraperitoneally (5 �l/g body wt) via a 27- gauge needle, once daily. Vehicle controls received an equivalent volume of 20% DMSO in PBS. Two-dimensional echocardiography and pulse wave Doppler ultrasound. Two-dimensional echocardiography and pulsed wave Doppler ultrasound was performed as a noninvasive method to assess pulmonary hemodynamics, using a Vivid 7 Advantage (GE Medical Systems, Milwaukee, WI) cardiovascular ultrasound machine with a small high frequency probe (I13L), as previously described (13). Briefly, rat pups were anesthetized with 5% (vol/vol) isoflurane, and the animal was laid supine while spontaneously breathing 2–3% (vol/vol) isoflurane through a modified face mask. A short axis view at the level of the aortic valve was obtained, and the pulmonary artery was identified using color flow Doppler. The pulsed Doppler gate was placed proximal to the pulmonary valve leaflets and aligned with an angle of insonation �20°, maximizing laminar flow. The RV ejection time (RVET) and pulmonary arterial acceleration time (PAAT) were measured using the pulmonary Doppler profile; RVET as the time from onset of systolic flow to completion of systolic flow, and PAAT from onset to peak pulmonary outflow velocity. A surrogate measure of PVR was calculated as a ratio of RVET to PAAT. To determine heart rate, measurements from three intersystolic intervals were av- eraged to account for beat-to-beat variability. Analyses were under- taken in a blinded fashion using an offline analysis system (ECHOPac, GE Medical Systems). Right ventricular hypertrophy. Measurement of RV hypertrophy using the Fulton index (RV/LV�S) is a well-established marker of PHT. The heart and lungs were separated, and the atria were removed inferior to the atrioventricular valves. The RV was separated from the left ventricle (LV) and septum. Each component was freeze dried overnight and weighed separately, as previously described (25). Measurement of systemic blood pressure. Systemic blood pressure was measured noninvasively using a tail cuff Doppler device (LE5002, Harvard Apparatus, Holliston, MA), as previously de- scribed (74). Rats were anesthetized with 5% (vol/vol) isoflurane; the animal was laid supine while spontaneously breathing 2–3% (vol/vol) isoflurane through a modified face mask. Due to technical limitations of the device in smaller animals, measurements were carried out between PND 21 and PND 26, as previously described (74). Exercise tolerance studies. Adult rats (10 wk of age) were trained daily on an Exer-3/6 animal treadmill (Columbus Instruments, Co- lumbus, OH) over a 7-day period. The treadmill speed was initially set to 10 m/min with a 10% uphill incline, and thereafter increased by increments of 2 m/min every 5 min up to a maximum speed of 20 m/min. Distance to fatigue was determined when the rat was unable to maintain pace with the treadmill belt for �3 s, despite encouragement to do so by tapping on the treadmill enclosure (electrical stimuli were not used). Following the 7-day training period, the final testing regimen was as follows: speed of 12.5 m/min for 1 min, followed by 15 m/min for 4 min, 17 m/min for 4 min, and 20 m/min until fatigue. Lowering of the hindquarters and a raised snout, resulting in an altered gait, generally precedes fatigue, which was confirmed by placing the animal on its back and observing a delayed (�2 s) righting reflex. In the case of a normal righting reflex, the test was repeated daily until fatigue was confirmed. Distance covered until fatigue was recorded to the nearest tenth of a meter. Tissue homogenate preparation. Right lower lobes (6 animals per group, equal sex distribution) were flash frozen, homogenized, and sonicated (40 W for 30 s) in RIPA cell lysis buffer [10 mM NaPO4, 0.3 M NaCl, 0.1% (wt/vol) sodium dodecyl sulfate (SDS), 1% (vol/vol) Nonidet P-40, 1% (vol/vol) sodium deoxycholate, 2 mM EDTA, pH 7.2] containing protease (Calbiochem) and phosphatase (Sigma Life Science) inhibitors. Left lung lobes (6 animals per group, equal sex distribution) were flash frozen, homogenized in lysis buffer (250 mM Tris·HCl, pH 8, 750 mM NaCl, 50 mM MgCl2, 5 mM EDTA, 5% Triton X-100) containing protease and phosphatase inhib- itors for immunoprecipitation. The lung homogenates were left on ice for 10 min before centrifugation (8,500 g for 10 min). The supernatant was collected and protein concentration was measured by Bradford assay. Samples were stored at �80°C until analysis. Serum collection. Following euthanasia of animals for tissue col- lection, the thoracic cavity was exposed and blood was drawn by direct LV cardiac puncture with a 27-gauge needle. Blood was kept at room temperature for 15 min to coagulate in microcentrifuge tubes. The tubes were spun down at 2,000 g for 10 min. Supernatant was collected by Pasteur pipette and stored at �80°C until assay. Western blot analyses. Lung homogenates from six animals per group (equal sex distribution) stored in RIPA buffer containing protease and phosphatase inhibitors. Tissue containing 50 –100 �g of protein was boiled for 5 min in SDS sample buffer [60 mM Tris·HCl, 10% (wt/vol) SDS, 5% (vol/vol) glycerol, 2 mM -mercaptoethanol, pH 6.8] and separated under reducing conditions by SDS polyacryl- amide gel electrophoresis for 1–2 h at 100 –150 V depending on protein of interest. Following electrophoresis, proteins were trans- ferred to PVDF membranes. All membranes were blocked with 5% BSA for 1 h at room temperature, followed by incubation with primary antibody overnight at 4°C. Blots were then washed with Tris-buffered saline with Tween 20 and placed in secondary antibody for 1 h at room temperature. Following blotting, bands were imaged using an enhanced chemiluminescence kit (SuperSignal West Dura Chemiluminescent Substrate, Thermo Scientific). Dilutions of anti- bodies were as follows: 1:1,000 for anti-RhoA (21 kDa), anti-cleaved L987SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. ROCK I (30 kDa), anti-P-Thr850 MYPT1 (80 kDa), anti-pan MYPT1 (135 kDa), anti-HIF-1� (120 kDa), and anti-eNOS (140 kDa); 1:2,000 for anti-ROCK I (180 kDa) and anti-ROCK II (180 kDa); 1:5,000 for anti-GAPDH (37 kDa); 1:10,000 for secondary antisera. Blots were electronically captured using a MicroChemi chemilu- minescent camera (DNR Bioimaging Systems, Jerusalem, Israel) and quantified by digital densitometry of nonsaturated images with back- ground density removed using ImageJ software (National Institutes of Health, Bethesda, MD). Quantitative analyses were conducted as follows: for each protein of interest, three gels were run comparing all samples from a group (normoxia vehicle, normoxia simvastatin, or hypoxia simvastatin) to all samples from the control group (hypoxia vehicle). Membranes were stripped and then reblotted for the normal- izer protein (e.g., pan-MYPT1, RhoA, or GAPDH) to account for differences in protein loading, and results expressed after normaliza- tion to the control band, as previously described (24). Representative images show 2 contiguous lanes (the same lanes for the protein of interest and the normalizer) from each group that best represented the quantitative analysis. RhoA activity assay. The concentration of lung tissue homogenates were equalized to 2 mg/ml in 1 ml lysis buffer. Anti-active RhoA monoclonal antibody (1 �l) was added to the tube followed by 30 �l of resuspended A/G agarose bead slurry. The tubes were incubated at 4°C for 1.5 h with gentle agitation following which beads were pelleted by centrifugation for 2 min at 6,500 g at 4°C. The supernatant was removed and stored for measurement of non-GTP-bound RhoA. The beads were washed three times with 0.5 ml 1 lysis buffer, centrifuging, aspirating, and discarding the supernatant each time. After the last wash, all supernatant was removed and discarded. The pellet was resuspended in 20 �l 2 reducing SDS-PAGE sample buffer. Samples were boiled for 5 min then centrifuged for 10 s at 6,500 g. Two Western blots were run in parallel with samples from the pellet or the supernatant. The membranes were probed with anti-RhoA primary antibody. The proportion of active RhoA was determined by band densities of active RhoA normalized to total RhoA [GTP-RhoA/ (GTP-RhoA�non-GTP-RhoA)]. Histological studies. Two males and two females were randomly selected from each of the experimental groups and euthanized by pentobarbital sodium overdose. Following the opening of the thoracic cavity and trachea cannulation, the pulmonary veins were divided. The pulmonary circulation was flushed with PBS and heparin, to clear the lungs of blood. The lungs were then perfusion fixed with parafor- maldehyde while air inflated at a constant pressure (20 cmH2O). Following dehydration and clearance in xylene, tissues were embed- ded in paraffin, cut into 5-�m sections, mounted onto Superfrost slides, allowed to air dry, and baked overnight at 43°C. For elastin staining, sections were dewaxed by immersion in xylene, rehydrated in ethanol, rinsed in several washes of distilled water, and then left overnight in Weigert’s resorcin-fuchsin stain. For �-smooth muscle actin immunostaining, concentration of primary antibody for �-smooth muscle actin was 1:1,500 and secondary goat anti-mouse antibody was 1:200 then counterstained with Carazzi hematoxylin. Slides were washed in tap water, dehydrated, and mounted with a coverslip using a 70% Permount/30% xylene solution, as previously described (25, 74). Preparation of cardiac tissue. Paraffin-embedded hearts, cut below the atrioventricular valves and oriented in the short axis, were cut into 5-�m sections and mounted onto Superfrost slides, allowed to air dry, and baked overnight at 43°C. Slides were dewaxed and rehydrated. Sections were stained with hematoxylin, washed in distilled water, and partially dehydrated before counterstaining with eosin. Slides were dehydrated and mounted with a coverslip using a 70% Per- mount/30% xylene solution. Whole sections were scanned by the Imaging Facility at the Hospital for Sick Children Research Institute, using a 3DHistech Pannoramic 250 Flash II Slide Scanner and Pannoramic Viewer 1.15.4 (3DHISTECH, Budapest, Hungary) soft- ware. Preparation of brain tissue. Paraffin-embedded left brain hemi- sphere oriented sagitally was cut into 5-�m sections just lateral to the ventricle and mounted onto Superfrost slides, allowed to air dry, and baked overnight at 43°C. Slides were dewaxed and rehydrated. Sec- tions were stained with Luxol fast blue solution (to identify myelin) overnight for 18 h at 56°C, washed in distilled water, placed in lithium carbonate solution for 30 s, and partially dehydrated before counter- staining with cresyl violet blue solution. Slides were dehydrated and mounted with a coverslip using a 70% Permount/30% xylene solution. Whole sections were scanned as described above. Measurement of percent medial wall area. Percent medial wall area (% MWA) was used as a marker of pulmonary vascular remodeling. Pulmonary arteries (20 –100 �m external diameter) were identified on elastin-stained sections by the presence of an inner and outer elastic lamina and proximity to respiratory bronchioles. A minimum of 40 arteries per animal from four unique sections were digitally captured (Pixera Penguin 600CL, San Jose, CA) and analysis was performed in a blinded fashion. Obliquely sectioned vessels where there was a greater than three times difference in perpendicular dimensions, were excluded. Using the “Quick Selection” tool (Adobe Photo- shop CS5, Adobe Systems, San Jose, CA), the area of the inner lumen and the whole vessel were outlined and pixel count deter- mined. The following formula was used to determine the % MWA: [(whole vessel area � inner luminal area)/whole vessel area] 100, as previously described (39). Evaluation of arterial muscularization. Degree of vessel muscular- ization was evaluated on sections stained for �-smooth muscle actin. Pulmonary arteries (20 –100 �m external diameter and proximity to respiratory bronchioles) were identified on �-smooth muscle actin- immunostained sections. A minimum of 40 arteries per animal from four unique sections were digitally captured and evaluated in a blinded fashion. Degree of muscularization was divided in to three categories: nonmuscular, partially muscular, or fully muscular based on the amount of brown stain surrounding each vessel (none, partially, or completely). Category of muscularity was expressed as a fraction of the total number of vessels examined. LC-MS/MS. Tissue samples weighing �200 mg were homogenized (750 mg/3 ml) in 1:1 2-propanol-100 mM ammonium bicarbonate. The equivalent of 225 mg (900 �l of homogenate) was added to a 1.7-ml plastic tube containing internal standards (Sigma-Aldrich, Oakville, Ontario, Canada) and briefly vortexed. A standard curve was generated (0.1–200 ng) for each analyte (mevalonic acid, FPP, and GGPP), treated in the same way as the tissue samples, but with 900 �l of 1:1 2-propanol-100 mM ammonium bicarbonate added instead of sample. Each tube was processed as follows: 900 �l of acetonitrile added, vortexed, chilled on ice, and then centrifuged for 10 min at 4°C at 20,000 g. The supernatant was transferred to a clean tube and the pellet was reextracted with 1 ml acetonitrile, vortexed, chilled on ice, and centrifuged for 10 min at 4°C at 20,000 g. The supernatants were combined and taken to dryness under a gentle flow of nitrogen gas. The residues were reconstituted in 50 �l of 7:3 methanol-ammonium hydroxide, transferred to 1.5 ml plastic tubes, and centrifuged for 10 min at 4°C at 20,000 g. The supernatant was transferred to a 200-�l insert in a 1-ml autosampler vial and injected for LC-MS/MS analysis. Samples were analyzed by LC-MS/MS using an Agilent 1290 HPLC with QTRAP 5500 mass spectrometer (AB Sciex, Concord, Ontario, Canada) in negative ion mode. One microliter of sample extract was injected into a Kinetex XB-C18 column (50 3.0 mm, 2.6 �m, Phenomenex) at a flow rate of 200 �l/min. Mevalonic acid, farnesyl pyrophosphate, and geranylgeranyl pyrophosphate were separated using a gradient of 5 mM ammonium bicarbonate and 0.05% triethylamine in water (MPA) and 5 mM ammonium bicarbonate and 0.05% triethyl- amine in 80% acetonitrile as follows: starting with 100% MPA, moving to 30% MPA from 0.6 to 4.0 min, then to 0% MPA from 4.0 to 5.0 min, 0% MPA was held from 5.0 to 7.5 min, after 7.5 min the system was returned to 100% MPA and allowed to equilibrate resulting in a total run time of 15 min. The following mass transitions (precursor ion L988 SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. to the product ion) were monitored for quantification in multiple reaction monitoring mode: mevalonic acid 147 to 59, farnesyl pyro- phosphate 381 to 79, and geranylgeranyl pyrophosphate 449 to 79. Data were analyzed and quantified using Analyst Software (AB Sciex). ELISA. Serum ALT, CK, LDL/VLDL cholesterol and total free cholesterol were measured according to the manufacturer’s protocols. Data presentation and statistical analysis. Values are expressed as means � SE. Table values are expressed as means � SD. Analyses were performed using Sigma Plot 12.5 (Systat Software, San Jose, CA). Statistical significance was determined by one-way ANOVA or Kruskal-Wallis ANOVA on ranks for data sets failing a normality test, followed by Student-Newman-Keuls post hoc test where significant (P � 0.05) intergroup differences were found. Fig. 2. Simvastatin decreased farnesyl pyro- phosphate and RhoA/ROCK activity in the hypoxia-exposed lung. Pups were exposed to chronic hypoxia (13% O2) or to normoxia (21% O2) from postnatal day 1 until day 14 (prevention) or day 21 (rescue). Pups were treated by daily intraperitoneal injections of simvastatin (2 mg/kg) or 20% DMSO in PBS (vehicle control) from birth to day 14 (A, B, and D), or from postnatal days 14 –21 (C and E). A: FPP lung isoprenoid interme- diates analyzed by LC-MS/MS. Western blot analyses of GTP-RhoA normalized to total RhoA (B and C) and pThr850-MYPT1 nor- malized to pan-MYPT1 (D and E). Repre- sentative immunoblots are shown below each graph with noncontiguous gel lanes demarcated by black lines. Values repre- sent means � SE for n � 5– 6 samples per group. *P � 0.05, by 1-way ANOVA on ranks (A) or 1-way ANOVA (B–D), com- pared with all other groups. #P � 0.001, by 1-way ANOVA, compared with all other groups. L989SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. RESULTS Lung FPP content and RhoA/ROCK activity were attenuated by simvastatin. Statins inhibit mevalonate synthesis, which reduces isoprenoid intermediate content (15, 37). As shown in Fig. 2A, chronic exposure to hypoxia had no effect on lung FPP content, compared with normoxia-exposed controls. Preven- tive treatment with simvastatin significantly reduced lung FPP content compared with hypoxia-exposed vehicle-treated pups (Fig. 2A). RhoA activity was measured by lung GTP-RhoA content, as a ratio of total (GTP�non-GTP bound) RhoA. Hypoxia-exposed, vehicle-treated pups had significantly ele- vated RhoA activity in the lung relative to normoxia controls at both PNDs 14 and 21 (Fig. 2, B and C). Lungs of chronic hypoxia-exposed pups treated with simvastatin had signifi- cantly reduced GTP-RhoA content when given as either pre- ventive or as rescue therapy (Figs. 2, B and C). Lung ROCK activity was determined by pThr850-MYPT1 content, as a ratio of the pan-MYPT1 protein. Lungs of hypoxia-exposed, vehi- cle-treated pups had significantly increased ROCK activity compared with normoxia controls at both PNDs 14 and 21 Fig. 3. Simvastatin prevented chronic hypoxia-induced pulmonary hypertension. Pups were exposed to chronic hypoxia (13% O2) to normoxia (21% O2) from postnatal day 1 until day 14. Pups were treated by daily intraperitoneal injections of simvastatin (2 mg/kg) or 20% DMSO in PBS (vehicle control). A: pulmonary vascular resistance (PVR) index, as measured by the right ventricular ejection time (RVET)-to-pulmonary arterial acceleration time (PAAT) ratio (values represent means � SE for n � 8 –12 animals/group). B: right ventricle (RV)-to-left ventricle � septum (LV�S) dry weight ratios (Fulton index) as a marker of right ventricular hypertrophy (values represent means � SE; n � 7–9 animals/group). C: tiled low-power photomicrographs of hematoxylin and eosin-stained cardiac sections, oriented in the short axis below the atrioventricular valves (RV, right ventricular cavity; scale bar � 2,000 �m), are shown to demonstrate differences in RV free wall thickness between groups. *P � 0.05, by 1-way ANOVA on ranks, compared with all other groups. L990 SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. (Fig. 2, D and E). Simvastatin significantly attenuated in- creased lung ROCK activity secondary to hypoxia, relative to the vehicle-treated animals, when given as either preventive or rescue therapy (Fig. 2, D and E). Simvastatin prevented chronic PHT. Similar to previous findings (74, 75), exposure to hypoxia significantly increased PVR index (Fig. 3A) and Fulton index (Fig. 3B) in vehicle- treated animals, compared with normoxia controls. Animals exposed to hypoxia and treated with simvastatin had signifi- cantly reduced PVR index (Fig. 3A) and Fulton index (Fig. 3B) compared with hypoxia-exposed vehicle-treated animals and had values comparable to normoxia controls. % MWA and degree of muscularization were used as measures of pulmonary arterial remodeling. As shown previously (22, 74, 75), and in Fig. 4A, pulmonary arteries of animals chronically exposed to hypoxia had greatly increased % MWA, which was accompa- nied by an increased proportion of fully muscularized arteries (Fig. 4C). Treatment with simvastatin reduced both % MWA (Fig. 4A) and proportion of fully muscularized arteries (Fig. 4C) relative to vehicle-treated controls. Chronic hypoxia-induced PHT was reversed and exercise capacity increased by rescue treatment with simvastatin. Pre- vious studies have demonstrated that systemic or inhaled treat- ment with a ROCK inhibitor reversed PHT induced by chronic Fig. 4. Simvastatin prevented chronic hypoxia-induced pulmonary arterial remodeling. Pups were exposed to chronic hypoxia (13% O2) or to normoxia (21% O2) from postnatal day 1 until day 14. Pups were treated by daily intraperitoneal injections of simvastatin (2 mg/kg) or 20% DMSO in PBS (vehicle control). A: percentage arterial medial wall area (values represent means � SE for n � 4 animals/group) as a marker of pulmonary arterial remodeling. B: representative photomicrographs of elastin staining (dark brown inner and outer elastic laminae delineating the medial vascular wall; scale bar � 50 �m) demonstrate differences between groups in medial wall thickening. C: degree of muscularization (values represent means � SE for n � 4 animals/group) as a marker of pulmonary arterial smooth muscle content. D: representative photomicrographs of �-smooth muscle actin staining (dark brown staining surrounding artery; scale bar � 50 �m) demonstrate proliferation of pulmonary arterial smooth muscle. *P � 0.05, by 1-way ANOVA on ranks compared with all other groups. #P � 0.05, by 1-way ANOVA compared with normoxia vehicle group for proportion of partially muscularized arteries. †P � 0.001, by 1-way ANOVA, compared with all other groups for proportions of nonmuscularized and ‡P � 0.05 for fully muscularized arteries. L991SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. hypoxia in newborn rats (22, 74). Similarly, relative to vehicle- treated animals, sustained rescue treatment with simvastatin significantly decreased PVR (Fig. 5A), RV hypertrophy (Fig. 5B), % MWA (Fig. 5C), and proportion of fully muscularized arteries (Fig. 5D) compared with chronic hypoxia-exposed vehicle-treated animals. Exercise capacity in chronic hypoxia, vehicle-treated rats was significantly reduced compared with air-exposed vehicle- or simvastatin-treated controls. Female rats ran a significantly greater distance compared with male rats across all groups and therefore data were stratified by sex. L992 SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. Rescue treatment with simvastatin significantly increased dis- tance run in females and males compared with hypoxia-ex- posed, vehicle-treated controls (Fig. 5E). Preventive treatment with simvastatin improved hypoxia- induced growth restriction and acute treatment with simvasta- tin did not affect systemic blood pressure. We have previously reported significant adverse effects of systemic ROCK inhibi- tion, including worsened hypoxia-induced growth restriction and greatly decreased systemic blood pressure (22, 75). As previously documented (75), chronic exposure to hypoxia from birth lead to a significant growth restriction (Fig. 6A). Preventive (Fig. 6A) treatment with simvastatin increased body weight at PND 14 compared with the hypoxia-ex- posed, vehicle-treated animals (Fig. 6A). Relative to nor- moxia-exposed controls, chronic hypoxia-exposed animals had significantly increased systolic blood pressure (Fig. 6B). Acute treatment with simvastatin had no effect on systolic blood pressure (Fig. 6B). Effects of hypoxia and preventive treatment with simvastatin on brain weight and periventricular myelin content. Relative to normoxia-exposed vehicle-treated animals, chronic exposure to hypoxia significantly decreased brain weight (Fig. 6C) and decreased periventricular myelin density (Fig. 6D). Treatment with simvastatin had no effects on brain weight in either normoxia- or hypoxia-exposed animals (Fig. 6C). Preventive treatment with simvastatin had no effect on periventricular myelin density in normoxia-exposed animals, while causing a significant increase in hypoxia-exposed animals (Fig. 6D). Preventive treatment with simvastatin did not increase se- rum markers of muscle or liver toxicity. Statin therapy has been reported in adult humans to cause myopathy (increased CK) and liver injury (increased ALT) (8). As shown in Table 1, neither chronic exposure to hypoxia nor treatment with simva- statin altered serum ALT or CK. In addition, no changes in these parameters were observed at 20 mg·kg�1·day�1, the highest dose employed in preliminary studies (data not shown). Preventive treatment with simvastatin had no effect on serum cholesterol. As shown in Table 1, neither chronic exposure to hypoxia nor treatment with simvastatin had any effect on total serum cholesterol. To control for the possible effects of DMSO vehicle on cholesterol levels (16), serum cholesterol was also measured in normoxia- and hypoxia- exposed animals not injected with vehicle. There was no difference in serum cholesterol between noninjected and vehicle-treated groups (data not shown). As shown in Table 1, chronic hypoxia significantly increased serum LDL cho- lesterol, which was unaffected by treatment with simvasta- tin. Effects of hypoxia and preventive treatment with simvastatin on lung cleaved ROCK I, ROCK I, ROCK II, HIF-1�, and eNOS contents. Chronic exposure to hypoxia greatly decreased cleaved ROCK I (constitutively active form of ROCK) content in the lung compared with normoxia-exposed controls (Fig. 7A). Normoxia-exposed animals treated with simvastatin had decreased cleaved ROCK I content compared with vehicle- treated animals (Fig. 7A). ROCK I content was significantly decreased by exposure to hypoxia, which was unaffected by treatment with simvastatin (Fig. 7A). There were no differences in ROCK II content between groups (Fig. 7C). HIF-1� is known to be upregulated by hypoxia. Accordingly, neonatal rat pups chronically exposed to hypoxia had significantly in- creased HIF-1� protein content in the lung compared with normoxia controls (Fig. 7D). Treatment with simvastatin had significantly reduced HIF-1� content in lungs exposed to chronic hypoxia (Fig. 7D). eNOS expression has been de- scribed to be attenuated by increased ROCK activity (64). As shown in Fig. 7E, preventive treatment with simvastatin in- creased eNOS content in both normoxia- and hypoxia-exposed animals. DISCUSSION We have previously established a critical role for the ROCK signaling in the pathogenesis of chronic hypoxic PHT in neonatal rats (22, 74, 75). In the present study, we observed that simvastatin both prevented and reversed chronic PHT, at least in part via modulation of RhoA and downstream ROCK activity. The novel aspect of this study was the examination of effects of simvastatin on chronic neonatal PHT. Despite favor- able results in adult experimental chronic PHT (21, 44, 52, 63), trials employing statins in human adults with chronic PHT have thus far shown an apparent lack of efficacy (57). The implications of these findings for translation of statins to the neonate remain open, given the potential for maturational differences in response to pharmacological agents that may affect the pulmonary vasculature. For example, selective sero- tonin reuptake inhibitors (SSRIs) impose a higher risk of PHT in newborns when given to mothers in late pregnancy (9), whereas studies in adults with chronic PHT suggest that SSRIs may improve outcome (30). A small observational study in children with chronic PHT showed improvement in hemody- namic parameters with simvastatin therapy (31). Importantly, the potential utility of statins as a means of targeting RhoA/ ROCK activity in the newborn likely extends beyond PHT. We have recently reported a contributory role for upregulated pulmonary ROCK signaling in experimental BPD-like lung Fig. 5. Simvastatin reversed chronic PHT in juveniles and improved exercise tolerance in young adults. Pups were exposed to chronic hypoxia (13% O2) or to normoxia (21% O2) from postnatal day 1 until day 21. Pups were treated by daily intraperitoneal injections of simvastatin (2 mg/kg) or 20% DMSO in PBS (vehicle control) from postnatal days 14 to 21. A: pulmonary vascular resistance index (PVRI), as measured by the right ventricular ejection time (RVET)-to-pulmonary arterial acceleration time (PAAT) ratio (values represent means � SE for n � 10 animals/group). B: right ventricle (RV)-to-left ventricle � septum (LV�S) dry weight ratios (Fulton index) as a marker of right ventricular hypertrophy (values represent means � SE n � 7–9 animals/group). C: percentage arterial medial wall area (values represent means � SE for n � 4 animals/group) as a marker of pulmonary arterial remodeling. D: degree of muscularization (values represent means � SE for n � 4 animals/group) as a marker of pulmonary arterial smooth muscle content. E: maximum exercise capacity measured in meters run (values represent means � SE for n � 5– 8 animals/group). *P � 0.05, by 1-way ANOVA on ranks, compared with all other groups. #P � 0.05, by 1-way ANOVA on ranks, compared with normoxia-exposed groups. †P � 0.05, by 1-way ANOVA, compared with all other groups, ‡P � 0.05, by 1-way ANOVA, compared with normoxia-exposed groups. §P � 0.05 (nonmuscularized arteries), �P � 0.001 (fully muscularized and partially muscularized arteries), by 1-way ANOVA, compared with hypoxia vehicle group. **P � 0.05, by 1-way ANOVA, compared with hypoxia vehicle group of the same sex. ††P � 0.001, by 1-way ANOVA, compared with hypoxia vehicle group of the same sex. L993SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. injury (39), a developmental lung disorder of prematurity characterized by inhibited pulmonary angiogenesis and alve- ologenesis. Similarly, pulmonary hypoplasia and vascular re- modeling in fetal rats with nitrofen-induced congenital dia- phragmatic hernia (CDH), in which RhoA may play a patho- logical role (67), was ameliorated by maternally administered simvastatin (45). As there are currently no effective preventive therapies for developmental lung disorders such and BPD and L994 SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. CDH, or to prevent or rescue chronic PHT in neonates (26), these findings point toward statins as a potentially useful therapy. We evaluated dose-response effects of simvastatin, establishing a dose that balanced efficacy and safety. This aspect is critical for potential translation since therapies de- signed to ameliorate injury to the lung and pulmonary vascu- lature could have unanticipated adverse effects on other sys- tems, particularly the developing brain. Collectively, our find- ings provide compelling initial evidence for the translational potential of statin therapy for neonatal pulmonary disease. We observed that preventively administered simvastatin attenuated the activity of RhoA, normalized PVR, and reduced both RV and pulmonary arterial structural remodeling. In our rescue protocol, the reduction of RhoA/ROCK activity also reduced PVR and led to significant but incomplete reversal of cardiac and vascular remodeling. Severity of pulmonary vas- cular changes secondary to chronic hypoxia peaks by 14 days of exposure and is stable from days 14 to 21; therefore, effects of rescue therapies (given from days 14 to 21) reflect reversal rather than slowed progression of injury. Rats exposed to chronic hypoxia receiving rescue treatment with simvastatin also had improved exercise capacity as young adults in keeping with an important and relevant functional improvement. The isoprenoid GGPP contributes to the pathogenesis of PHT by prenylation-mediated intracellular trafficking and membrane localization, and subsequent activation of RhoA (21, 47, 60, 72). In the present study, we observed an increase in FPP and in HIF-1� content in the lungs of chronic hypoxia- exposed rats. We also observed that simvastatin reduced lung content of FPP in hypoxia-exposed animals, with a trend toward reduction in normoxia controls. Presumably, a reduc- tion of FPP also led to decreased GGPP as indicated by reduced RhoA activity; however, we were unable to reliably measure GGPP content in lung. It is known that HIF-1� activity is elevated in hypoxia, which in turn increases tran- scription of RhoA and/or ROCK (53). Treatment with simva- statin decreased pulmonary HIF-1� levels in chronic hypoxia- exposed animals but did not affect lung content of RhoA or of either ROCK isoform (20, 69). Therefore, the effect of simva- statin could be largely attributed to downregulation of RhoA activity via inhibition of isoprenoid intermediates, rather than by changes in protein content mediated by HIF-1�. In support of this observation, Girgis and colleagues (21) demonstrated that concurrent supplementation of mevalonate with simvasta- tin treatment negated any beneficial effects in experimental PHT. The pathology of chronic hypoxic PHT is characterized in neonatal animals by severe pulmonary vascular remodeling (71). In previous studies, inhibition of ROCK signaling pre- vented vascular remodeling by inhibiting SMC proliferation (75). Rescue therapy with ROCK inhibitor opposed the anti- apoptotic effect of ROCK (specifically by ROCK II), therefore stimulating SMC apoptosis and leading to reversal of remod- eling (74). Inhibited NO-cGMP signaling plays a major role in the pathogenesis of chronic PHT (32, 49). ROCK inhibits eNOS activity and disrupts the stability of eNOS mRNA, thus reducing NO bioavailability (17, 28). In the present study, we observed that simvastatin increased eNOS content, which fur- ther supports suppression of ROCK activity as a major mech- anism for simvastatin effects on chronic neonatal PHT (35). Neonates are particularly susceptible to adverse and off- target effects of pharmacological agents (2). Documented ad- verse effects of statins in adult humans include hepatic injury and muscle pain. In rabbits, high-dose simvastatin (50 mg·kg�1·day�1) increased serum lactate dehydrogenase, CK, and ALT (27). In the present study, we did not observe any significant increase in serum levels of ALT or CK, even at doses of simvastatin as high as 20 mg/kg. Systemically admin- istered ROCK inhibitors restrict somatic growth, thus limiting translational potential to the neonate (75). In the present study, simvastatin did not adversely affect somatic growth in nor- moxia-exposed animals and actually restored normal growth in chronic hypoxia-exposed animals. In addition, simvastatin did not affect systolic blood pressure, unlike systemic ROCK inhibitors (22). We did not examine effects of exposure to hypoxia or of simvastatin therapy on RhoA/ROCK activity in systemic vessels, but we speculate that a lack of effect of simvastatin on systemic vascular tone reflects a greater hypox- ia-induced upregulation of RhoA/ROCK activity in the pulmo- nary, compared with the systemic, vasculature. The usual target of statin therapy is a reduction in serum cholesterol, which in the neonate may represent an adverse Fig. 6. Effects of chronic hypoxia from birth and preventive simvastatin treatment on somatic and brain growth, brain myelination, and systemic blood pressure. Pups were exposed to chronic hypoxia (13% O2) or to normoxia (21% O2) from postnatal day 1 until day 14. Pups were treated by daily intraperitoneal injections of simvastatin (2 mg/kg) or 20% DMSO in PBS (vehicle control). A: body weight. Values represent means � SE for n � 10 –12 animals per group. B: systemic blood pressure following therapy with simvastatin. Values represent means � SE for n � 6 animals per group. C: brain weight. Values represent means � SE for n � 6 samples per group. D: myelin pixel density. Values represent means � SE for n � 4 samples per group. E: tiled low-power representative photomicrographs of Luxol fast blue staining on sagittal brain sections (myelin � dark blue stain; scale bar � 2,500 �m). *P � 0.05, by 1-way ANOVA, compared with all other groups. #P � 0.05, by 1-way ANOVA, compared with normoxia controls. †P � 0.05, by 1-way ANOVA, compared with normoxia vehicle. ‡P � 0.001, by 1-way ANOVA, compared with normoxia controls. §P � 0.05, by 1-way ANOVA, compared with hypoxia vehicle group. Table 1. Effects of preventative treatment with simvastatin (2 mg·kg�1·day�1) on serum markers of toxicity Serum Analyte Normoxia Vehicle Normoxia Simvastatin Hypoxia Vehicle Hypoxia Simvastatin ALT, �mol/ml 1.112 � 0.276 1.277 � 0.242 1.126 � 0.271 1.373 � 0.407 CK, �mol/ml 10.169 � 0.774 10.328 � 1.261 10.66 � 0.483 10.811 � 0.286 Total cholesterol, mg/dl 447.44 � 108.63 537.524 � 125.892 499.514 � 115.439 439.539 � 109.911 LDL cholesterol, mg/dl 273.112 � 49.38 252.61 � 38.923 387.556 � 103.269* 362.039 � 53.775# Values represent means � SD for n � 6 animals per group. *P � 0.05, by 1-way ANOVA, compared to both Normoxia groups. #P � 0.05, by 1-way ANOVA, compared to Normoxia Simvastatin group. L995SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. Fig. 7. Effects of preventive simvastatin on lung cleaved ROCK I, ROCK I, ROCK II, HIF-1�, and eNOS content. Pups were exposed to chronic hypoxia (13% O2) or to normoxia (21% O2) from postnatal day 1 until day 14. Pups were treated by daily intraperitoneal injections of simvastatin (2 mg/kg) or 20% DMSO in PBS (vehicle control). Western blot analyses for cleaved ROCK I (A), ROCK I (B), ROCK II (C), HIF-1� (D), and eNOS (E), all normalized to GAPDH. Representative immunoblots are shown with noncontiguous gel lanes demarcated by black lines. Values represent means � SE for n � 5– 6 samples per group. *P � 0.05, by 1-way ANOVA on ranks, compared with all other groups. #P � 0.05, by 1-way ANOVA on ranks, compared with hypoxia simvastatin group. †P � 0.05, by 1-way ANOVA, compared with normoxia simvastatin group. ‡P � 0.001, by 1-way ANOVA, compared with all other groups. §P � 0.05, by 1-way ANOVA, compared with hypoxia vehicle group. L996 SIMVASTATIN PREVENTS AND REVERSES CHRONIC NEONATAL PHT AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00345.2016 • www.ajplung.org Downloaded from journals.physiology.org/journal/ajplung at Carnegie Mellon Univ (128.182.081.034) on April 5, 2021. effect, given the reliance of the developing brain on cholesterol for normal development (55). Interestingly, neither simvastatin nor the DMSO vehicle altered total serum cholesterol levels or LDL cholesterol in the newborn rat. This finding is likely explained by the fact that breastfeeding neonates rely less on endogenous synthesis of cholesterol to maintain serum levels, due to the presence of high levels of cholesterol in breast milk (19). Indeed, high doses of statins have not been found to alter brain cholesterol levels, which is required for myelin formation (1, 10, 46, 68). Lipid soluble statins such as simvastatin can cross the blood-brain barrier by lactonization. However, statins do not accumulate in the brain, being rapidly eliminated by the P-glycoprotein transporter (51). Furthermore, CYP3A4, the enzyme responsible for simvastatin metabolism and activation, is absent from the rodent brain (61, 68) and has only been detected in the human brain at one-tenth the level of liver CYP3A4 (73). Prematurely born humans have an increased risk for long-term neurodevelopmental disability. In models of newborn neurological injury and abnormal development, st- atins prevented injury through modulation of inflammation, leading to normalized morphology, white matter content, and brain weight (43, 54, 70). While there were no reported adverse effects from simvastatin in a small observational study in children with PHT, further study is needed to assess safety and efficacy in the newborn population despite our present reas- suring findings (31). There are a number of limitations to this study. First, our chronic hypoxia model of PHT does not mimic chronic PHT secondary to developmental lung disorders, such as BPD, which is among the most common causes of chronic PHT in neonates and infants (26). Therefore, effects of statin therapy should be studied in models mimicking inhibited pulmonary angiogenesis and arrested lung development, as observed in BPD (50). Second, use of simvastatin as a rescue therapy showed incomplete effects compared with prevention, which may have been overcome by prolongation of therapy. Third, we did not conduct functional neurological examination to substantiate our morphological findings suggesting an im- provement in hypoxia-mediated disruption of myelination with simvastatin treatment. Fourth, our focus was restricted to the activity of RhoA. Many other GTPase proteins could be similarly affected by statin therapy, based on their dependence for prenylation-mediated activation. Of note, the Ras family of G proteins and other members of the Rho family such as Rac1 and Cdc42 may warrant further investigation since they are known to be involved in cardiac hypertrophy, actin cytoskel- eton remodeling, and SMC proliferation (29, 65). Lastly, eval- uation of sex differences in response to therapy is a relevant biological variable that should be evaluated in future studies. In summary, simvastatin-mediated inhibition of isoprenoid intermediates downregulates RhoA activity, leading to preven- tion and reversal of chronic PHT. Our findings suggest that simvastatin was well tolerated, lacked apparent adverse sys- temic or neurological effects, and therefore holds promise as a potentially translatable means of limiting pathological ROCK activity in the neonate. ACKNOWLEDGMENTS The authors thank Denis Reynaud and Ashley St. Pierre of the Analytical Facility for Bioactive Molecules, The Hospital for Sick Children, Toronto, Canada, for assistance with LC- 550 MS/MS analysis of lung tissue isoprenoid intermediates. GRANTS This work was supported by operating funding from the Canadian Institutes of Health Research (CIHR; MOP-84290 to R. P. Jankov) and by infrastructure funding from the Canada Foundation for Innovation (to R. P. Jankov). A. Jain was supported by a Clinician-Scientist Training Program Award from the Hospital for Sick Children Research Training Centre and by a Queen Elizabeth II/Heart and Stroke Foundation of Ontario Graduate Scholarship in Science and Technology. M. J. Wong was supported by a Graduate Scholarship from the Department of Physiology, University of Toronto and by a Lorne Phenix Graduate award from the Cardiovascular Sciences Collaborative Program, Faculty of Medicine, University of Toronto. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS M.J.W. and R.P.J. conceived and designed research; M.J.W., C.K., J.I., A.J., and R.P.J. performed experiments; M.J.W., A.J., and R.P.J. analyzed data; M.J.W. and R.P.J. interpreted results of experiments; M.J.W. and R.P.J. prepared figures; M.J.W. and R.P.J. drafted manuscript; M.J.W., C.K., J.I., A.J., and R.P.J. edited and revised manuscript; M.J.W., C.K., J.I., A.J., and R.P.J. approved final version of manuscript. REFERENCES 1. Adibhatla RM, Hatcher JF. Altered lipid metabolism in brain injury and disorders. Subcell Biochem 49: 241–268, 2008. 2. Allegaert K, van den Anker J. Neonatal drug therapy: the first frontier of therapeutics for children. Clin Pharmacol Ther 98: 288 –297, 2015. 3. Ambalavanan N, Mourani P. Pulmonary hypertension in bronchopul- monary dysplasia. Birth Defects Res A Clin Mol Teratol 100: 240 –246, 2014. 4. 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