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Sildenafil Prevents Marfan-Associated Emphysema and Early Pulmonary Artery Dilation in Mice

2019; Elsevier BV; Volume: 189; Issue: 8 Linguagem: Inglês

10.1016/j.ajpath.2019.05.003

1525-2191

Zoe White, Nadia Milad, Arash Y. Tehrani, Jennifer Lamothe, James C. Hogg, Mitra Esfandiarei, Michael A. Seidman, Steven Booth, Tillie‐Louise Hackett, Mathieu C. Morissette, Pascal Bernatchez,

Connective tissue disorders research

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in fibrillin-1 (Fbn1). Although aortic rupture is the major cause of mortality in MFS, patients also experience pulmonary complications, which are poorly understood. Loss of basal nitric oxide (NO) production and vascular integrity has been implicated in MFS aortic root disease, yet their contribution to lung complications remains unknown. Because of its capacity to potentiate the vasodilatory NO/cyclic guanylate monophosphate signaling pathway, we assessed whether the phosphodiesterase-5 inhibitor, sildenafil (SIL), could attenuate aortic root remodeling and emphysema in a mouse model of MFS. Despite increasing NO-dependent vasodilation, SIL unexpectedly elevated mean arterial blood pressure, failed to inhibit MFS aortic root dilation, and exacerbated elastic fiber fragmentation. In the lung, early pulmonary artery dilation observed in untreated MFS mice was delayed by SIL treatment, and the severe emphysema-like alveolar destruction was prevented. In addition, improvements in select parameters of lung function were documented. Subsequent microarray analyses showed changes to gene signatures involved in the inflammatory response in the MFS lung treated with SIL, without significant down-regulation of connective tissue or transforming growth factor-β signaling genes. Because phosphodiesterase-5 inhibition leads to improved lung histopathology and function, the effects of SIL against emphysema warrant further investigation in the settings of MFS despite limited efficacy on aortic root remodeling. Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in fibrillin-1 (Fbn1). Although aortic rupture is the major cause of mortality in MFS, patients also experience pulmonary complications, which are poorly understood. Loss of basal nitric oxide (NO) production and vascular integrity has been implicated in MFS aortic root disease, yet their contribution to lung complications remains unknown. Because of its capacity to potentiate the vasodilatory NO/cyclic guanylate monophosphate signaling pathway, we assessed whether the phosphodiesterase-5 inhibitor, sildenafil (SIL), could attenuate aortic root remodeling and emphysema in a mouse model of MFS. Despite increasing NO-dependent vasodilation, SIL unexpectedly elevated mean arterial blood pressure, failed to inhibit MFS aortic root dilation, and exacerbated elastic fiber fragmentation. In the lung, early pulmonary artery dilation observed in untreated MFS mice was delayed by SIL treatment, and the severe emphysema-like alveolar destruction was prevented. In addition, improvements in select parameters of lung function were documented. Subsequent microarray analyses showed changes to gene signatures involved in the inflammatory response in the MFS lung treated with SIL, without significant down-regulation of connective tissue or transforming growth factor-β signaling genes. Because phosphodiesterase-5 inhibition leads to improved lung histopathology and function, the effects of SIL against emphysema warrant further investigation in the settings of MFS despite limited efficacy on aortic root remodeling. Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder (CTD) caused by mutations in the fibrillin-1 (Fbn1) gene.1Dietz H.C. Cutting G.R. Pyeritz R.E. Maslen C.L. Sakai L.Y. Corson G.M. Puffenberger E.G. Hamosh A. Nanthakumar E.J. Curristin S.M. Marfan syndrome caused by a recurrent de novo missense mutation in the fibrillin gene.Nature. 1991; 352: 337-339Crossref PubMed Scopus (1600) Google Scholar Fibrillin-1 is a part of the extracellular matrix (ECM) and acts as a major structural component of elastic fibers in large arteries. Loss of fibrillin-1 integrity leads to degeneration of elastic fibers, which predisposes vessels to microdissections, aneurysms, and eventual vessel rupture.2Pyeritz R.E. The Marfan syndrome.Annu Rev Med. 2000; 51: 481-510Crossref PubMed Scopus (333) Google Scholar In addition, MFS patients experience significant pulmonary complications associated with degradation of the lung parenchyma, including emphysematous-like tissue destruction, spontaneous pneumothorax, upper airway collapsibility, and pulmonary artery trunk dilation.3Ishii H. Shima R. Kinoshita Y. Kushima H. Marfan syndrome presenting with diffuse emphysematous change of the lung.BMJ Case Rep. 2018; 2018 (bcr-2017-224056)Google Scholar, 4Lee J.-J. Galatioto J. Rao S. Ramirez F. Costa K.D. Losartan attenuates degradation of aorta and lung tissue micromechanics in a mouse model of severe Marfan syndrome.Ann Biomed Eng. 2016; 44: 2994-3006Crossref PubMed Scopus (22) Google Scholar, 5Uriarte J.J. Meirelles T. Gorbenko Del Blanco D. Nonaka P.N. Campillo N. Sarri E. Navajas D. Egea G. Farre R. Early impairment of lung mechanics in a murine model of Marfan syndrome.PLoS One. 2016; 11: e0152124Crossref PubMed Scopus (17) Google Scholar Currently, there are no MFS-specific options to prevent or attenuate the severity of lung emphysema and associated remodeling in this population as pharmacotherapy generally focuses on improving aortic root stability. The two main medications for the prophylactic management of aortic complications are as follows: losartan, an angiotensin II type 1 receptor blocker capable of reducing afterload, blood pressure (BP), and pathogenic transforming growth factor (TGF)-β signaling; and atenolol, a β-adrenoreceptor blocker that lowers heart rate and pulse wave velocity. Despite encouraging preclinical data, the overall efficacy of these medications at slowing the rate of aortic root widening and reducing all-cause mortality is controversial.6Lacro R.V. Dietz H.C. Sleeper L.A. Yetman A.T. Bradley T.J. Colan S.D. Pearson G.D. Selamet Tierney E.S. Levine J.C. Atz A.M. Benson D.W. Braverman A.C. Chen S. De Backer J. Gelb B.D. Grossfeld P.D. Klein G.L. Lai W.W. Liou A. Loeys B.L. Markham L.W. Olson A.K. Paridon S.M. Pemberton V.L. Pierpont M.E. Pyeritz R.E. Radojewski E. Roman M.J. Sharkey A.M. Stylianou M.P. Wechsler S.B. Young L.T. Mahony L. 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Pulmonary vascular abnormalities in chronic obstructive pulmonary disease undergoing lung transplant.J Heart Lung Transplant. 2013; 32: 1262-1269Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar, 11Dinh-Xuan A.T. Higenbottam T.W. Clelland C.A. Pepke-Zaba J. Cremona G. Butt A.Y. Large S.R. Wells F.C. Wallwork J. Impairment of endothelium-dependent pulmonary-artery relaxation in chronic obstructive lung disease.N Engl J Med. 1991; 324: 1539-1547Crossref PubMed Scopus (478) Google Scholar Nitric oxide (NO) is one of the key vasodilatory mediators often found to be down-regulated in endothelial dysfunction. It can be released constitutively by both the vascular endothelium and the lung epithelium,12Pechkovsky D.V. Zissel G. Goldmann T. Einhaus M. Taube C. Magnussen H. Schlaak M. Muller-Quernheim J. Pattern of NOS2 and NOS3 mRNA expression in human A549 cells and primary cultured AEC II.Am J Physiol Lung Cell Mol Physiol. 2002; 282: L684-L692Crossref PubMed Scopus (47) Google Scholar resulting in blunted smooth muscle contractility and attenuated vascular and bronchial tone (Sandoo et al13Sandoo A. van Zanten J.J.C.S.V. Metsios G.S. Carroll D. Kitas G.D. The endothelium and its role in regulating vascular tone.Open Cardiovasc Med J. 2010; 4: 302-312Crossref PubMed Scopus (447) Google Scholar and Ricciardolo14Ricciardolo F.L.M. Multiple roles of nitric oxide in the airways.Thorax. 2003; 58: 175-182Crossref PubMed Scopus (268) Google Scholar). However, a more enigmatic and ill-defined aspect of the biology of NO is its capacity to protect underlying tissues from inflammation and pathologic remodeling.15Kubes P. Suzuki M. Granger D.N. Nitric oxide: an endogenous modulator of leukocyte adhesion.Proc Natl Acad Sci U S A. 1991; 88: 4651-4655Crossref PubMed Scopus (2811) Google Scholar, 16Sharma A. Sellers S. Stefanovic N. Leung C. Tan S.M. Huet O. Granville D.J. Cooper M.E. de Haan J.B. Bernatchez P. Direct endothelial nitric oxide synthase activation provides atheroprotection in diabetes-accelerated atherosclerosis.Diabetes. 2015; 64: 3937-3950Crossref PubMed Scopus (49) Google Scholar Aberrant endothelial function has been reported in MFS patients,17Wilson D.G. Bellamy M.F. Ramsey M.W. Goodfellow J. Brownlee M. Davies S. Wilson J.F. Lewis M.J. Stuart A.G. Endothelial function in Marfan syndrome: selective impairment of flow-mediated vasodilation.Circulation. 1999; 99: 909-915Crossref PubMed Scopus (58) Google Scholar which could take part in the severe remodeling typically observed in their aorta and pulmonary artery trunk, a process akin to accelerated arteriolar aging.18Salvi P. Grillo A. Marelli S. Gao L. Salvi L. Viecca M. Di Blasio A.M. Carretta R. Pini A. Parati G. Aortic dilatation in Marfan syndrome: role of arterial stiffness and fibrillin-1 variants.J Hypertens. 2018; 36: 77-84Crossref PubMed Scopus (19) Google Scholar Interestingly, our research group has shown that losartan and atenolol differ in their effect on endothelial function in MFS tissues and that losartan-induced increase in NO bioavailability is responsible for its anti-aortic root remodeling effects.19Chung A.W.Y. Au Yeung K. Cortes S.F. Sandor G.G.S. Judge D.P. Dietz H.C. van Breemen C. Endothelial dysfunction and compromised eNOS/Akt signaling in the thoracic aorta during the progression of Marfan syndrome.Br J Pharmacol. 2007; 150: 1075-1083Crossref PubMed Scopus (80) Google Scholar, 20Sellers S.L. Milad N. Chan R. Mielnik M. Jermilova U. Huang P.L. de Crom R. Hirota J.A. Hogg J.C. Sandor G.G. Van Breemen C. Esfandiarei M. Seidman M.A. Bernatchez P. Inhibition of Marfan syndrome aortic root dilation by losartan: role of angiotensin II receptor type 1-independent activation of endothelial function.Am J Pathol. 2018; 188: 574-585Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Because of the key similarities between vascular and pulmonary structures, it is plausible that increased NO signaling could attenuate MFS-associated lung pathology. The predominant secondary messenger of NO that initiates vasodilation is phosphodiesterase-5 (PDE5), an enzyme abundantly expressed in the pulmonary vasculature that degrades cGMP.21Butrous G. The role of phosphodiesterase inhibitors in the management of pulmonary vascular diseases.Glob Cardiol Sci Pract. 2014; 2014: 257-290PubMed Google Scholar The PDE5 inhibitor, sildenafil (SIL), can potentiate NO-induced vasodilation,22Kass D.A. Takimoto E. Nagayama T. Champion H.C. Phosphodiesterase regulation of nitric oxide signaling.Cardiovasc Res. 2007; 75: 303-314Crossref PubMed Scopus (125) Google Scholar decrease pulmonary vascular resistance and bronchodilation, and improve the 6-minute walk distance in patients with pulmonary arterial hypertension (Barnett et al23Barnett C.F. Machado R.F. Sildenafil in the treatment of pulmonary hypertension.Vasc Health Risk Manag. 2006; 2: 411-422Crossref PubMed Scopus (115) Google Scholar). In addition, SIL was found to preserve or even improve ventilation-perfusion matching, a marker of impaired lung perfusion and oxygenation, in interstitial lung disease,24Lederer D.J. Bartels M.N. Schluger N.W. Brogan F. Jellen P. Thomashow B.M. Kawut S.M. Sildenafil for chronic obstructive pulmonary disease: a randomized crossover trial.COPD. 2012; 9: 268-275Crossref PubMed Scopus (68) Google Scholar as well as improve pulmonary hemodynamics and exercise tolerance in patients with chronic obstructive pulmonary disease–associated pulmonary arterial hypertension.25Blanco I. Gimeno E. Munoz P.A. Pizarro S. Gistau C. Rodriguez-Roisin R. Roca J. Barbera J.A. Hemodynamic and gas exchange effects of sildenafil in patients with chronic obstructive pulmonary disease and pulmonary hypertension.Am J Respir Crit Care Med. 2010; 181: 270-278Crossref PubMed Scopus (225) Google Scholar, 26Galie N. Ghofrani H.A. Torbicki A. Barst R.J. Rubin L.J. Badesch D. Fleming T. Parpia T. Burgess G. Branzi A. Grimminger F. Kurzyna M. Simonneau G. Sildenafil citrate therapy for pulmonary arterial hypertension.N Engl J Med. 2005; 353: 2148-2157Crossref PubMed Scopus (2048) Google Scholar, 27Pepke-Zaba J. Gilbert C. Collings L. Brown M.C.J. Sildenafil improves health-related quality of life in patients with pulmonary arterial hypertension.Chest. 2008; 133: 183-189Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 28Singh T.P. Rohit M. Grover A. Malhotra S. Vijayvergiya R. A randomized, placebo-controlled, double-blind, crossover study to evaluate the efficacy of oral sildenafil therapy in severe pulmonary artery hypertension.Am Heart J. 2006; 151: 851.e1-851.e5Crossref PubMed Scopus (214) Google Scholar Hence, amplification of NO signaling via PDE5 inhibition can promote lung tissue homeostasis. Whether this is caused by simple dilation of the pulmonary arterial tree or a more complex vasodilation-independent antiremodeling effect is unknown. Thus, the aim of this study was to determine the therapeutic potential of PDE5 inhibition on the vascular and pulmonary complications of MFS syndrome in a preclinical model. We describe how treatment with SIL can prevent emphysematous-like destruction of the lung parenchyma and improve respiratory mechanics despite a limited anti-aortic root remodeling effect in the C1039G model of MFS. Our data suggest that potentiation of cGMP-dependent NO signaling with SIL could help attenuate pathologic lung remodeling in settings of MFS and perhaps other CTDs. Male and female Marfan mice harboring the Fbn1 C1039G+/- mutation (MFS; position 1041 in mice) and littermate control [wild-type (WT); Fbn1 C1039G+/+] mice were bred and housed in the Genetically Engineered Models facility at the Centre for Heart Lung Innovation, University of British Columbia (Vancouver, BC, Canada). All animals were housed on a standard 12-hour light/dark cycle, were fed standard laboratory chow (LabDiet #5001; LabDiet, St. Louis, MO), and were maintained using breeding and procedures approved by the University of British Columbia Animal Care Committee. Sildenafil citrate (Pfizer, New York, NY) treatment was provided via drinking water (50 mg/kg per day) starting at 6 weeks of age and continuing until 24 weeks. Dosage was titrated to body weight and volume of water consumed per cage (averaged per day). Water was replaced, and doses were adjusted every week for the first 4 weeks of treatment and every 2 weeks thereafter. Control animals consumed regular water. Experiments were performed in two different cohorts, 6 to 7 months apart, with all values combined unless specified otherwise. Echocardiographic measurements were performed on anesthetized mice (0.75% v/v isoflurane, 1.5 L O2) using a VisualSonics Vevo 2100 system (Fujifilm, Tokyo, Japan) with an MS-550D 40-MHz probe by a technician blinded to genotype and treatment group. Aortic root measurements were averaged from multiple measurements taken in both M- and B-modes at the level of the sinus of Valsalva, as previously described.4Lee J.-J. Galatioto J. Rao S. Ramirez F. Costa K.D. Losartan attenuates degradation of aorta and lung tissue micromechanics in a mouse model of severe Marfan syndrome.Ann Biomed Eng. 2016; 44: 2994-3006Crossref PubMed Scopus (22) Google Scholar, 20Sellers S.L. Milad N. Chan R. Mielnik M. Jermilova U. Huang P.L. de Crom R. Hirota J.A. Hogg J.C. Sandor G.G. Van Breemen C. Esfandiarei M. Seidman M.A. Bernatchez P. Inhibition of Marfan syndrome aortic root dilation by losartan: role of angiotensin II receptor type 1-independent activation of endothelial function.Am J Pathol. 2018; 188: 574-585Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Data for treated and untreated mice were analyzed at both 12 and 24 weeks of age. Systemic BP was noninvasively measured before sacrifice (at 24 weeks) using the tail cuff system (CODA2; Kent Scientific, Torrington, CT). Briefly, mice were lightly anesthetized (0.75% v/v isoflurane, 1.5 L O2) and placed on a warming tray with the tail inserted into an inflatable cuff, where systolic BP and diastolic BP were measured. Mean arterial pressure (MAP) was calculated as follows: (1/3 × systolic BP) + (2/3 × diastolic BP). Mice were euthanized at 24 weeks of age under terminal anesthesia (5% v/v isoflurane, 1.5 L O2), followed by cervical dislocation. The heart, lungs, and ascending aorta were excised; separated from the descending aorta distal to the left subclavian artery; and fixed in 10% neutral buffer formalin. Lungs were dissected away from the hearts, as per standard methods, across the vasculature and airways. Hearts were cut away from the aorta via a section through the basal ventricles parallel to the atrioventricular plane, and both segments were embedded cut side down in paraffin. Sections were analyzed every 50 μm through the aortic root. As previously described,20Sellers S.L. Milad N. Chan R. Mielnik M. Jermilova U. Huang P.L. de Crom R. Hirota J.A. Hogg J.C. Sandor G.G. Van Breemen C. Esfandiarei M. Seidman M.A. Bernatchez P. Inhibition of Marfan syndrome aortic root dilation by losartan: role of angiotensin II receptor type 1-independent activation of endothelial function.Am J Pathol. 2018; 188: 574-585Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar elastic fiber integrity was assessed at the sinus of Valsalva on slides stained with Movat's pentachrome by a cardiac pathologist blinded to genotype and treatment status (M.S.). Elastic fiber fragmentation was scored on a scale of 0 to 3, wherein 0 indicates no fragmentation; 1, mild fragmentation (0% to 20%); 2, moderate fragmentation (20% to 40%); and 3, extensive fragmentation (>40%). Aortic root medial thickness was quantified on histologic sections using Aperio ImageScope software version 11.2.0.780 (Leica Biosystems, Nussloch, Germany). After separating from the heart, the right lung was placed in RNAlater (Thermo Fisher Scientific, Waltham, MA) and stored at −20°C until required. Histology of the left lung was performed, as previously described, via inflation, paraffin embedding, and staining of sections with Masson's trichrome; then, airspace enlargement was analyzed by mean linear intercept.20Sellers S.L. Milad N. Chan R. Mielnik M. Jermilova U. Huang P.L. de Crom R. Hirota J.A. Hogg J.C. Sandor G.G. Van Breemen C. Esfandiarei M. Seidman M.A. Bernatchez P. Inhibition of Marfan syndrome aortic root dilation by losartan: role of angiotensin II receptor type 1-independent activation of endothelial function.Am J Pathol. 2018; 188: 574-585Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar Serial lung sections stained with hematoxylin and eosin were used to visualize cellular aggregates. Lung function was assessed in a separate cohort of mice (aged 24 weeks) using the FlexiVent (SCIREQ, Montréal, QC, Canada). Mice were anesthetized with 100 mg/kg ketamine and 10 mg/kg xylazine, tracheotomized with an 18-gauge blunted needle, and mechanically ventilated at a respiratory rate of 150 breaths/minute and a tidal volume of 10 mL/kg, with a pressure limit of 30 cm H2O. Muscle paralysis was achieved using pancuronium (2 mg/kg; Sandoz, Boucherville, QC, Canada) to prevent respiratory efforts during the measurement. The following sequence of measures was repeated three times and averaged for analysis using FlexiWare Software version 7.6 (SCIREQ): deep inflation, Snapshot-150, Quick Prime-3, and pressure/volume-loop to obtain measures for compliance, resistance, tissue damping, and lung inspiratory capacity, respectively. As previously described,29Lechasseur A. Jubinville E. Routhier J. Berube J.-C. Hamel-Auger M. Talbot M. Lamothe J. Aubin S. Pare M.-E. Beaulieu M.-J. Bosse Y. Duchaine C. Morissette M.C. Exposure to electronic cigarette vapors affects pulmonary and systemic expression of circadian molecular clock genes.Physiol Rep. 2017; 5 (e13440)Crossref PubMed Scopus (30) Google Scholar RNA from lung samples stored in RNAlater at −20°C was extracted using the RNeasy Plus Universal Mini kit (number 73404; Qiagen, Hilden, Germany), following manufacturer's instructions. RNA levels and purity were assessed using the NanoVue spectrophotometer (GE Health Care, Chicago, IL), and RNA degradation was assessed using gel electrophoresis. Whole transcript expression of RNA samples (n = 4 to 6 for each group) was determined using the GeneChip Clariom S Assay HT mouse (Affymetrix) by Genome Québec (Montréal, QC, Canada). Microarray data analyses were performed using R statistical software version 1.1.463 using the R packages affy, samr, and limma (R Core Team, Vienna, Austria). Data are available at http://www.ncbi.nlm.nih.gov (accession number GSE128481). The computational algorithm (ImmuCC)30Newman A.M. Liu C.L. Green M.R. Gentles A.J. Feng W. Xu Y. Hoang C.D. Diehn M. Alizadeh A.A. Robust enumeration of cell subsets from tissue expression profiles.Nat Methods. 2015; 12: 453-457Crossref PubMed Scopus (5000) Google Scholar was used to predict the tissue composition of 25 immune cells in untreated and treated WT and MFS lungs using the microarray expression data generated above. The 25 immune cell types were grouped into the 15 major cell types for visualization.30Newman A.M. Liu C.L. Green M.R. Gentles A.J. Feng W. Xu Y. Hoang C.D. Diehn M. Alizadeh A.A. Robust enumeration of cell subsets from tissue expression profiles.Nat Methods. 2015; 12: 453-457Crossref PubMed Scopus (5000) Google Scholar The algorithm was performed using R statistical software version 3.5.3. A workflow schematic of this process has been added in Supplemental Figure S1. The descending thoracic aorta was dissected from the thoracic cage and cleaned of fat and connective tissue in ice-cold Krebs solution (118 mmol/L NaCl, 22.5 mmol/L NaHCO3, 4 mmol/L KCl, 1.2 mmol/L NaH2PO4, 2 mmol/L CaCl2, 2 mmol/L MgCl2, 11 mmol/L dextrose, and 0.01 mmol/L ibuprofen). Segments of the thoracic aorta (2 mm thick) were mounted isometrically in a small vessel myograph (AS Danish Myotechnology, Aarhus, Denmark) to measure smooth muscle cell (SMC) contractility. Thoracic aorta segments were left to equilibrate for 30 minutes at 37°C in Krebs solution, aerated continuously with 95% O2/5% CO2. Vessels were stretched to the optimal tension (6.0 mN) for 30 minutes, as previously described,19Chung A.W.Y. Au Yeung K. Cortes S.F. Sandor G.G.S. Judge D.P. Dietz H.C. van Breemen C. Endothelial dysfunction and compromised eNOS/Akt signaling in the thoracic aorta during the progression of Marfan syndrome.Br J Pharmacol. 2007; 150: 1075-1083Crossref PubMed Scopus (80) Google Scholar and thereafter challenged twice with 30 mmol/L KCl to determine the viability and reactivity of aortic SMCs, followed by a concentration-dependent dose-response with phenylephrine (3 nmol/L to 100 μmol/L). Percentage contraction was calculated as the percentage increase or decrease in force with respect to untreated WT mice, where the maximum recorded response was arbitrarily set to 100% contraction. To study the effect of NO on SMC contractility, vessels were incubated with Nω-nitro-l-arginine methyl ester (L-NAME; 200 mmol/L), a NO inhibitor, for 30 minutes before addition of phenylephrine (3 nmol/L to 100 μmol/L). Statistical analyses and table figures were prepared using GraphPad Prism Software version 6.01 (GraphPad Software, San Diego, CA). Two-way analysis of variance was used to compare the means between each genotype and treatment grouping. Sidak's post-hoc test was used to correct for multiple comparisons. Data are presented as the means ± SEM, unless otherwise specified, with P < 0.05 considered significant. For analysis of the gene expression data, only genes with a false discovery rate of 1.2 or <0.8 were considered statistically significant. In accordance with previously published data,20Sellers S.L. Milad N. Chan R. Mielnik M. Jermilova U. Huang P.L. de Crom R. Hirota J.A. Hogg J.C. Sandor G.G. Van Breemen C. Esfandiarei M. Seidman M.A. Bernatchez P. Inhibition of Marfan syndrome aortic root dilation by losartan: role of angiotensin II receptor type 1-independent activation of endothelial function.Am J Pathol. 2018; 188: 574-585Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar it was observed by echocardiography that 12-week–old MFS mice exhibit significantly larger pulmonary artery and aortic root diameters compared with WT controls in the absence of treatment (P < 0.01) (Figure 1, A and C). At 24 weeks, untreated WT and MFS pulmonary artery diameters did not significantly differ (Figure 1B), whereas MFS mice still showed larger aortic root diameters compared with WT controls (Figure 1D). The significant difference in pulmonary artery diameter at 12 weeks was mitigated by SIL treatment, but there was no significant effect on pulmonary artery diameter at 24 weeks or on aortic root diameter at either 12 or 24 weeks (Figure 1, A–D). Due to a lack of anti-aortic root remodeling effects, BP and heart function were also investigated. Chronic SIL treatment in MFS mice led to an unexpected but statistically significant increase in systemic systolic BP and diastolic BP (P < 0.05) (Figure 1E) as well as mean arterial blood pressure (MAP) (Supplemental Figure S2A), an effect not observed in WT mice. Further echocardiographic evaluation demonstrated increased stroke volume and left ventricular diastolic diameter in untreated MFS mice when compared with WT controls, with no effect on other measured parameters (P < 0.05) (Supplemental Table S1). SIL treatment had no effect on left ventricular heart function (Supplemental Table S1), suggesting both a pulmonary artery–specific effect and increased afterload, which could rationalize a lack of anti-aortic root widening effect of SIL on MFS tissues. After euthanasia, histology of aortic root tissues revealed that MFS mice treated with SIL exhibit greater elastic fiber fragmentation and medial thickening when compared with untreated MFS mice (Figure 2, A–C). Aortic root sections showed robust PDE5 staining throughout medial SMC layers (Figure 2D) by immunochemistry. Ex vivo myography using aortic tissues confirmed a potentiation of vasodilatory NO signaling by SIL, as quantified by decreased KCl-induced contractility (Supplemental Figure S2B) and attenuated phenylephrine-induced contractility in an L-NAME–sensitive manner (Figure 2, E–G). These ex vivo data confirm the unabated increase in elastic fiber degradation and medial thickening during the aortic root widening process in MFS mice treated with SIL despite potentiated vasodilatory NO signaling, which suggests that the anti-aortic root remodeling properties of NO are vasodilation and PDE5 independent. C1039G MFS mice present with an emphysematous phenotype (Figure 3A). A 35% greater mean linear intercept was observed in untreated MFS mice compared with WT controls, a marker of increased airspace widening (P < 0.001) (Figure 3, A and B). WT mice did not show a significant effect of SIL on mean linear intercept (Figure 3B), whereas MFS mice showed a significantly lower mean linear intercept in response to SIL compared with untreated MFS mice (Figure 3, B and C). Because of the apparent anti-emphysematous effect of SIL, lung function experiments were performed in an additional cohort of mice (Figure 4). Consistent with profound emphysema, pressure-volume loop dynamics, including compliance, resistance, tissue damping, lung inspiratory capacity (Figure 4, A–E), hysteresis, and elastance (data not shown), were negatively affected in pathogenic MFS lungs. In MFS lung, SIL significantly reduced pulmonary compliance (a measure of elastic tissue distensibility) by 17% (P < 0.05) and prevented MFS-associated changes in resistance (Figure 4, B and C). Although tissue damping, a measure of resistance and alveolar energy dissipation, was unaffected by SIL treatment (Figure 4D), total lung capacity was mildly improved (13%), albeit not statistically si