Severe Pulmonary Arterial Hypertension Is Characterized by Increased Neutrophil Elastase and Relative Elafin Deficiency
2021; Elsevier BV; Volume: 160; Issue: 4 Linguagem: Inglês
10.1016/j.chest.2021.06.028
ISSN1931-3543
AutoresAndrew J. Sweatt, Kazuya Miyagawa, Christopher J. Rhodes, Shalina Taylor, Patricia A. del Rosario, Andrew Hsi, François Haddad, Edda Spiekerkoetter, Michal Bental-Roof, Richard D. Bland, Emilia M. Swietlik, Stefan Gräf, Martin R. Wilkins, Nicholas W. Morrell, Mark R. Nicolls, Marlene Rabinovitch, Roham T. Zamanian,
Tópico(s)Cardiovascular Issues in Pregnancy
ResumoBackgroundPreclinical evidence implicates neutrophil elastase (NE) in pulmonary arterial hypertension (PAH) pathogenesis, and the NE inhibitor elafin is under early therapeutic investigation.Research QuestionAre circulating NE and elafin levels abnormal in PAH and are they associated with clinical severity?Study Design and MethodsIn an observational Stanford University PAH cohort (n = 249), plasma NE and elafin levels were measured in comparison with those of healthy control participants (n = 106). NE and elafin measurements were then related to PAH clinical features and relevant ancillary biomarkers. Cox regression models were fitted with cubic spline functions to associate NE and elafin levels with survival. To validate prognostic relationships, we analyzed two United Kingdom cohorts (n = 75 and n = 357). Mixed-effects models evaluated NE and elafin changes during disease progression. Finally, we studied effects of NE-elafin balance on pulmonary artery endothelial cells (PAECs) from patients with PAH.ResultsRelative to control participants, patients with PAH were found to have increased NE levels (205.1 ng/mL [interquartile range (IQR), 123.6-387.3 ng/mL] vs 97.6 ng/mL [IQR, 74.4-126.6 ng/mL]; P < .0001) and decreased elafin levels (32.0 ng/mL [IQR, 15.3-59.1 ng/mL] vs 45.5 ng/mL [IQR, 28.1-92.8 ng/mL]; P < .0001) independent of PAH subtype, illness duration, and therapies. Higher NE levels were associated with worse symptom severity, shorter 6-min walk distance, higher N-terminal pro-type brain natriuretic peptide levels, greater right ventricular dysfunction, worse hemodynamics, increased circulating neutrophil levels, elevated cytokine levels, and lower blood BMPR2 expression. In Stanford patients, NE levels of > 168.5 ng/mL portended increased mortality risk after adjustment for known clinical predictors (hazard ratio [HR], 2.52; CI, 1.36-4.65, P = .003) or prognostic cytokines (HR, 2.63; CI, 1.42-4.87; P = .001), and the NE level added incremental value to established PAH risk scores. Similar prognostic thresholds were identified in validation cohorts. Longitudinal NE changes tracked with clinical trends and outcomes. PAH PAECs exhibited increased apoptosis and attenuated angiogenesis when exposed to NE at the level observed in patients' blood. Elafin rescued PAEC homeostasis, yet the required dose exceeded levels found in patients.InterpretationBlood levels of NE are increased while elafin levels are deficient across PAH subtypes. Higher NE levels are associated with worse clinical disease severity and outcomes, and this target-specific biomarker could facilitate therapeutic development of elafin. Preclinical evidence implicates neutrophil elastase (NE) in pulmonary arterial hypertension (PAH) pathogenesis, and the NE inhibitor elafin is under early therapeutic investigation. Are circulating NE and elafin levels abnormal in PAH and are they associated with clinical severity? In an observational Stanford University PAH cohort (n = 249), plasma NE and elafin levels were measured in comparison with those of healthy control participants (n = 106). NE and elafin measurements were then related to PAH clinical features and relevant ancillary biomarkers. Cox regression models were fitted with cubic spline functions to associate NE and elafin levels with survival. To validate prognostic relationships, we analyzed two United Kingdom cohorts (n = 75 and n = 357). Mixed-effects models evaluated NE and elafin changes during disease progression. Finally, we studied effects of NE-elafin balance on pulmonary artery endothelial cells (PAECs) from patients with PAH. Relative to control participants, patients with PAH were found to have increased NE levels (205.1 ng/mL [interquartile range (IQR), 123.6-387.3 ng/mL] vs 97.6 ng/mL [IQR, 74.4-126.6 ng/mL]; P < .0001) and decreased elafin levels (32.0 ng/mL [IQR, 15.3-59.1 ng/mL] vs 45.5 ng/mL [IQR, 28.1-92.8 ng/mL]; P < .0001) independent of PAH subtype, illness duration, and therapies. Higher NE levels were associated with worse symptom severity, shorter 6-min walk distance, higher N-terminal pro-type brain natriuretic peptide levels, greater right ventricular dysfunction, worse hemodynamics, increased circulating neutrophil levels, elevated cytokine levels, and lower blood BMPR2 expression. In Stanford patients, NE levels of > 168.5 ng/mL portended increased mortality risk after adjustment for known clinical predictors (hazard ratio [HR], 2.52; CI, 1.36-4.65, P = .003) or prognostic cytokines (HR, 2.63; CI, 1.42-4.87; P = .001), and the NE level added incremental value to established PAH risk scores. Similar prognostic thresholds were identified in validation cohorts. Longitudinal NE changes tracked with clinical trends and outcomes. PAH PAECs exhibited increased apoptosis and attenuated angiogenesis when exposed to NE at the level observed in patients' blood. Elafin rescued PAEC homeostasis, yet the required dose exceeded levels found in patients. Blood levels of NE are increased while elafin levels are deficient across PAH subtypes. Higher NE levels are associated with worse clinical disease severity and outcomes, and this target-specific biomarker could facilitate therapeutic development of elafin. FOR EDITORIAL COMMENT, SEE PAGE 1177Pulmonary arterial hypertension (PAH) is a progressive occlusive arteriopathy that often culminates in right heart failure and death, despite available therapies. Persistent inflammation is a well-recognized feature of PAH,1Rabinovitch M. Guignabert C. Humbert M. Nicolls M.R. Inflammation and immunity in the pathogenesis of pulmonary arterial hypertension.Circ Res. 2014; 115: 165-175Crossref PubMed Scopus (498) Google Scholar,2Huertas A. Perros F. Tu L. et al.Immune dysregulation and endothelial dysfunction in pulmonary arterial hypertension: a complex interplay.Circulation. 2014; 129: 1332-1340Crossref PubMed Scopus (110) Google Scholar arising from aberrant reparative immune responses that follow a disease-provoking vascular insult.3Tamosiuniene R. Tian W. Dhillon G. et al.Regulatory T cells limit vascular endothelial injury and prevent pulmonary hypertension.Circ Res. 2011; 109: 867-879Crossref PubMed Scopus (181) Google Scholar,4Nicolls M.R. Voelkel N.F. The roles of immunity in the prevention and evolution of pulmonary arterial hypertension.Am J Respir Crit Care Med. 2017; 195: 1292-1299Crossref PubMed Scopus (43) Google Scholar Extracellular matrix breakdown accompanies this inflammation, propagating immune cell activation and recruitment.5Humbert M. Morrell N.W. Archer S.L. et al.Cellular and molecular pathobiology of pulmonary arterial hypertension.J Am Coll Cardiol. 2004; 43: 13S-24SCrossref PubMed Scopus (1239) Google Scholar,6Thenappan T. Chan S.Y. Weir E.K. Role of extracellular matrix in the pathogenesis of pulmonary arterial hypertension.Am J Physiol Heart Circ Physiol. 2018; 315: H1322-H1331Crossref PubMed Scopus (57) Google Scholar Levels of perivascular immune cells and blood cytokines correlate with PAH clinical severity,7Savai R. Pullamsetti S.S. Kolbe J. et al.Immune and inflammatory cell involvement in the pathology of idiopathic pulmonary arterial hypertension.Am J Respir Crit Care Med. 2012; 186: 897-908Crossref PubMed Scopus (209) Google Scholar, 8Stacher E. Graham B.B. Hunt J.M. et al.Modern age pathology of pulmonary arterial hypertension.Am J Respir Crit Care Med. 2012; 186: 261-272Crossref PubMed Scopus (381) Google Scholar, 9Soon E. Holmes A.M. Treacy C.M. et al.Elevated levels of inflammatory cytokines predict survival in idiopathic and familial pulmonary arterial hypertension.Circulation. 2010; 122: 920-927Crossref PubMed Scopus (465) Google Scholar, 10Cracowski J.L. Chabot F. Labarere J. et al.Proinflammatory cytokine levels are linked to death in pulmonary arterial hypertension.Eur Respir J. 2014; 43: 915-917Crossref PubMed Scopus (77) Google Scholar suggesting that inflammation may contribute to disease progression. FOR EDITORIAL COMMENT, SEE PAGE 1177 Neutrophils are among the immune cells involved and release neutrophil elastase (NE), a protease implicated in PAH arteriopathy.11Taylor S. Dirir O. Zamanian R.T. Rabinovitch M. Thompson A.A.R. The role of neutrophils and neutrophil elastase in pulmonary arterial hypertension.Front Med (Lausanne). 2018; 5: 217Crossref PubMed Scopus (32) Google Scholar Neutrophils isolated from patients with PAH have enhanced NE release capacity12Rose F. Hattar K. Gakisch S. et al.Increased neutrophil mediator release in patients with pulmonary hypertension—suppression by inhaled iloprost.Thromb Haemost. 2003; 90: 1141-1149PubMed Google Scholar: NE localizes to pulmonary arterial smooth muscle cells and neointimal lesions in PAH lungs,13Kim Y.M. Haghighat L. Spiekerkoetter E. et al.Neutrophil elastase is produced by pulmonary artery smooth muscle cells and is linked to neointimal lesions.Am J Pathol. 2011; 179: 1560-1572Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar and NE activity temporally associates with vascular remodeling in experimental pulmonary hypertension.14Zhu L. Wigle D. Hinek A. et al.The endogenous vascular elastase that governs development and progression of monocrotaline-induced pulmonary hypertension in rats is a novel enzyme related to the serine proteinase adipsin.J Clin Invest. 1994; 94: 1163-1171Crossref PubMed Scopus (92) Google Scholar NE is thought to trigger remodeling via extracellular matrix degradation, which causes release of growth factors, clustering and activation of their receptors, and ensuing migration and proliferation of smooth muscle cells and fibroblasts.15Thompson K. Rabinovitch M. Exogenous leukocyte and endogenous elastases can mediate mitogenic activity in pulmonary artery smooth muscle cells by release of extracellular-matrix bound basic fibroblast growth factor.J Cell Physiol. 1996; 166: 495-505Crossref PubMed Scopus (120) Google Scholar, 16Tu L. Dewachter L. Gore B. et al.Autocrine fibroblast growth factor-2 signaling contributes to altered endothelial phenotype in pulmonary hypertension.Am J Respir Cell Mol Biol. 2011; 45: 311-322Crossref PubMed Scopus (95) Google Scholar, 17Cowan K.N. Jones P.L. Rabinovitch M. Elastase and matrix metalloproteinase inhibitors induce regression, and tenascin-C antisense prevents progression, of vascular disease.J Clin Invest. 2000; 105: 21-34Crossref PubMed Scopus (246) Google Scholar NE may perpetuate inflammation by generating chemotactic elastin fragments,18Senior R.M. Griffin G.L. Mecham R.P. Chemotactic responses of fibroblasts to tropoelastin and elastin-derived peptides.J Clin Invest. 1982; 70: 614-618Crossref PubMed Scopus (118) Google Scholar proteolytically modifying cytokines19Alfaidi M. Wilson H. Daigneault M. et al.Neutrophil elastase promotes interleukin-1beta secretion from human coronary endothelium.J Biol Chem. 2015; 290: 24067-24078Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar,20Valenzuela-Fernandez A. Planchenault T. Baleux F. et al.Leukocyte elastase negatively regulates stromal cell-derived factor-1 (SDF-1)/CXCR4 binding and functions by amino-terminal processing of SDF-1 and CXCR4.J Biol Chem. 2002; 277: 15677-15689Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar and downregulating bone morphogenetic protein receptor 2 (BMPR2) signaling.21Burton V.J. Ciuclan L.I. Holmes A.M. Rodman D.M. Walker C. Budd D.C. Bone morphogenetic protein receptor II regulates pulmonary artery endothelial cell barrier function.Blood. 2011; 117: 333-341Crossref PubMed Scopus (89) Google Scholar,22Li W. Hoenderos K. Salmon R.M. et al.Neutrophil and redox dependent proteolysis of bone morphogenetic protein 9: potential role in the pathogenesis of pulmonary arterial hypertension.Thorax. 2013; 68 (A146-A146)Crossref PubMed Google Scholar Therapies that antagonize NE have yielded favorable preclinical results.23Cowan K.N. Heilbut A. Humpl T. Lam C. Ito S. Rabinovitch M. Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor.Nat Med. 2000; 6: 698-702Crossref PubMed Scopus (309) Google Scholar We found that recombinant elafin, an endogenously produced NE inhibitor, reversed experimental pulmonary hypertension and caused neointimal lesions to regress in cultured lung tissue.24Nickel N.P. Spiekerkoetter E. Gu M. et al.Elafin reverses pulmonary hypertension via caveolin-1-dependent bone morphogenetic protein signaling.Am J Respir Crit Care Med. 2015; 191: 1273-1286Crossref PubMed Scopus (90) Google Scholar Elafin seems to amplify BMPR2 signaling and to promote normal angiogenesis.24Nickel N.P. Spiekerkoetter E. Gu M. et al.Elafin reverses pulmonary hypertension via caveolin-1-dependent bone morphogenetic protein signaling.Am J Respir Crit Care Med. 2015; 191: 1273-1286Crossref PubMed Scopus (90) Google Scholar,25Sa S. Gu M. Chappell J. et al.Induced pluripotent stem cell model of pulmonary arterial hypertension reveals novel gene expression and patient specificity.Am J Respir Crit Care Med. 2017; 195: 930-941Crossref PubMed Scopus (54) Google Scholar Based on these findings, we launched a phase 1 trial of elafin therapy for PAH (ClinicalTrials.gov Identifier: NCT03522935). An increasing number of novel PAH therapies have failed in clinical trials, despite promising preclinical data. Experts call for the development of biomarkers relevant to pathobiological features, clinical disease state, and drug mechanism during initial investigation phases to inform later-phase clinical trial design.26Sitbon O. Gomberg-Maitland M. Granton J. et al.Clinical trial design and new therapies for pulmonary arterial hypertension.Eur Respir J. 2019; 53: 1801908Crossref PubMed Scopus (81) Google Scholar,27Grieve A.P. Chow S.C. Curram J. et al.Advancing clinical trial design in pulmonary hypertension.Pulm Circ. 2013; 3: 217-225Crossref PubMed Scopus (14) Google Scholar Despite preclinical evidence linking NE to PAH pathobiological features and the therapeutic potential of elafin, no study has evaluated circulating NE and elafin as clinical biomarkers. As part of the bench-to-bedside development of elafin therapy, we aimed to determine whether NE and elafin blood levels are abnormal in PAH and are associated with disease severity and outcomes. We hypothesized that NE levels would be increased and elafin levels would be deficient across PAH subtypes, with more pronounced derangements in severe disease. Plasma NE and elafin levels were measured in an observational cohort study of patients with PAH and healthy control participants enrolled at Stanford University (Stanford, CA). Levels of these biomarkers (1) were evaluated across PAH subtypes in comparison with those of control participants, (2) were related to PAH clinical features and outcomes, (3) were reassessed over time to examine changes during disease progression, and (4) were correlated with ancillary blood measures (leukocyte subsets, cytokines, and BMPR2 expression). To validate observed relationships with outcomes, we analyzed existing multicenter United Kingdom (UK) data from prior PAH proteome studies. Guided by the NE and elafin blood levels found in patients, we then assessed the effects of NE-elafin imbalance on pulmonary arterial endothelial cells (PAECs) isolated from PAH lung explants. The primary cohort included patients with World Health Organization Group 1 PAH that underwent evaluation at Stanford and had blood collected for the Vera Moulton Wall Center biobank between 2008 and 2013 (n = 249). The study was approved by the Stanford University Institutional Review Board (protocol no., 14083), and all participants provided informed consent. PAH was diagnosed according to existing guidelines and required mean pulmonary arterial pressure (mPAP) of ≥ 25 mm Hg, pulmonary vascular resistance of > 240 dynes/sec/cm5, and wedge pressure of ≤ 15 mm Hg.28Simonneau G. Gatzoulis M.A. Adatia I. et al.Updated clinical classification of pulmonary hypertension.J Am Coll Cardiol. 2013; 62: D34-D41Crossref PubMed Scopus (1917) Google Scholar We excluded patients with chronic lung disease, left ventricular systolic dysfunction, valvular disease, chronic infection, or any acute illness within 1 month (e-Fig 1). Control participants (n = 106) were included from the Stanford Healthy Aging Population Study, which screened volunteers for cardiovascular and immune health from 2009 through 2013 (e-Appendix 1). Internal jugular venous samples were collected during the index right heart catheterization at Stanford, regardless of whether PAH was incident or prevalent. Plasma samples were processed immediately and stored under strict protocols (e-Appendix 2A). We obtained follow-up samples from a patient subset during routine surveillance catheterization (n = 70). NE and elafin levels were measured by enzyme-linked immunoabsorbent assay (Hycult Biotech) (e-Appendix 3A). The Stanford Pulmonary Hypertension Database was used to extract patient demographics, clinical features, and background therapies at the time of plasma collection. In patients with follow-up sampling, therapy interventions and clinical trends were captured. All patients were observed over time for the outcome of death or lung transplantation (e-Appendix 2B). For patients with same-day data available from prior Vera Moulton Wall Center biobank studies,29Sweatt A.J. Hedlin H.K. Balasubramanian V. et al.Discovery of distinct immune phenotypes using machine learning in pulmonary arterial hypertension.Circ Res. 2019; 124: 904-919Crossref PubMed Scopus (69) Google Scholar,30Sweatt A. Wells R. Purington N. et al.BMPR2 expression is reduced in blood across PAH subtypes but does not reflect disease severity.Am J Respir Crit Care Med. 2018; 197: A2449Google Scholar we obtained blood measurements of leukocyte subset counts (CBC differential), 48 cytokines (BioPlex multiplex immunoassay; Bio-Rad), and BMPR2 messenger RNA expression (real-time quantitative reverse transcriptase polymerase chain reaction analysis; Applied Biosystems) (e-Appendix 3B, 3C). Patients with idiopathic PAH were included from two UK studies: a prior published cohort31Rhodes C.J. Wharton J. Ghataorhe P. et al.Plasma proteome analysis in patients with pulmonary arterial hypertension: an observational cohort study.Lancet Respir Med. 2017; 5: 717-726Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar enrolled at Imperial College from 2002 through 2011 (cohort A; n = 75) and a multicenter cohort recruited from 2014 through 2018 for the UK National Cohort Study of Idiopathic and Heritable PAH (cohort B; n = 357). Aptamer-based peripheral plasma NE measurements (SomaScan; Somalogic) and survival data were obtained (e-Appendix 4). Explanted lungs were procured from PAH transplant recipients (n = 3) and control donors (n = 3) through the Pulmonary Hypertension Breakthrough Initiative. Harvested PAECs were cultured and treated with various elastase and elafin dose combinations (e-Table 1). Under each treatment condition, we evaluated PAEC apoptosis (caspase 3/7 assay) and angiogenesis activity (tube formation) (e-Appendix 5).24Nickel N.P. Spiekerkoetter E. Gu M. et al.Elafin reverses pulmonary hypertension via caveolin-1-dependent bone morphogenetic protein signaling.Am J Respir Crit Care Med. 2015; 191: 1273-1286Crossref PubMed Scopus (90) Google Scholar,32Diebold I. Hennigs J.K. Miyagawa K. et al.BMPR2 preserves mitochondrial function and DNA during reoxygenation to promote endothelial cell survival and reverse pulmonary hypertension.Cell Metab. 2015; 21: 596-608Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar Analyses were performed using R version 3.5.1 software (R Foundation for Statistical Computing) (e-Appendix 6). NE and elafin levels in PAH were compared with those of control participants via Mann-Whitney U test. Receiver operating characteristic curves assessed the PAH discriminatory power of each biomarker, and ideal discrimination cutoffs were identified (Youden's statistic). To evaluate univariate biomarker relationships with clinical features, we applied the Spearman ρ statistic (continuous variables), the Mann-Whitney U test (binary variables), the Kruskal-Wallis test (categorical variables), and the Cuzick test (ordinal variables). We also fit median regression models adjusted for age, sex, and BMI to associate NE and elafin levels with disease severity metrics. Biomarker changes were related to clinical features over time via mixed-effects models. NE and elafin levels were correlated with leukocyte subset counts and BMPR2 expression levels in blood. Significance analysis of microarrays was implemented to ascertain differentially expressed cytokines among patients with increased NE levels. The outcome of time to death or transplantation from blood sampling was analyzed, and Kaplan-Meier transplant-free survival estimates were compared across biomarker quantiles. Univariate Cox regression models were fitted to assess the relationship between each biomarker and the outcome. When the assumption of linearity between biomarker levels and log hazard was not satisfied, Cox models were fitted with cubic spline functions to examine nonlinear relationships. Bootstrapped spline model estimates were used to identify the biomarker threshold beyond which mortality risk remained significantly increased. This prognostic threshold was evaluated in multivariate Cox models that adjusted for known predictors of PAH risk, including clinical parameters and cytokines. We also determined whether the prognostic threshold added incremental value to established PAH risk stratification scores. The Registry to Evaluate Early and Long-Term PAH Disease Management 2.0 calculator,33Benza R.L. Gomberg-Maitland M. Elliott C.G. et al.Predicting survival in patients with pulmonary arterial hypertension: the REVEAL risk score calculator 2.0 and comparison with ESC/ERS-based risk assessment strategies.Chest. 2019; 156: 323-337Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar the French Pulmonary Hypertension Registry algorithm,34Boucly A. Weatherald J. Savale L. et al.Risk assessment, prognosis and guideline implementation in pulmonary arterial hypertension.Eur Respir J. 2017; 50: 1700889Crossref PubMed Scopus (302) Google Scholar and the Comparative Prospective Registry of Newly Initiated Therapies for Pulmonary Hypertension score35Hoeper M.M. Kramer T. Pan Z. et al.Mortality in pulmonary arterial hypertension: prediction by the 2015 European pulmonary hypertension guidelines risk stratification model.Eur Respir J. 2017; 50: 1700740Crossref PubMed Scopus (280) Google Scholar stratified low-, intermediate-, and high-risk groups (e-Appendix 6C). The predictive performance of risk score-only and biomarker-inclusive Cox regression models was compared by likelihood ratio test. The Stanford cohort with PAH (Table 1) had a median age of 49 years (interquartile range [IQR], 38-59 years) and was predominantly female (76.7%). The most common PAH subtypes were connective tissue disease associated (31.7%) and idiopathic (27.7%), followed by drugs and toxins associated (18.9%), congenital heart disease associated (15.3%), and portopulmonary hypertension (6.4%). Many participants showed New York Heart Association functional class III or IV symptoms (57.0%), and PAH was hemodynamically severe: mPAP of 50 mm Hg (IQR, 40-60 mm Hg) and pulmonary vascular resistance 821 dynes/sec/cm5 (IQR, 506-1197 dynes/sec/cm5). Most patients were either treatment naïve (35.0%) or were receiving monotherapy (28.1%).Table 1Stanford Cohort Characteristics at Baseline Measurement of NE and ElafinVariableControl Participants (N = 106)All PAH (n = 249)PAH Subgroup With Follow-up NE and Elafin Measurements (n = 70)Age, y58 (48-72)49 (38-59)50 (40-58)Sex Female58 (54.7)191 (76.7)53 (75.7) Male48 (45.3)58 (23.3)17 (24.3)PAH cause IPAH + HPAHaHPAH: n = 3 patients (1.2% of cohort), who had confirmed BMPR2 mutations. Mutation status was not otherwise evaluated routinely in the cohort.—69 (27.7)20 (28.6) D&T-associated PAH—47 (18.9)17 (24.3) CTD-associated PAH—79 (31.7)18 (25.7) PoPH—16 (6.4)7 (10.0) CHD-associated PAH—38 (15.3)8 (11.4)NYHA FC I—14 (5.6)5 (7.1) II—93 (37.3)33 (47.1) III—114 (45.8)24 (34.3) IV—28 (11.2)8 (11.4)6MWD, m—423 (341-513)433 (366-512)Therapy extent Naïve—87 (35.0)19 (27.1) Monotherapy—70 (28.1)21 (30.0) Dual therapy—69 (27.7)24 (34.3) Triple therapy—23 (9.2)6 (8.6)Therapy class PDE-5 inhibitor—119 (47.8)36 (51.4) ERA—74 (29.7)22 (31.4) Prostacyclin—84 (33.7)29 (41.4)Dlco, % predicted—71 (54-87)73 (64-89)NT-proBNP, pg/mL—289 (80-1238)216 (75-934)Hemodynamics Right atrial pressure, mm Hg—7 (5-11)7 (4-12) mPAP, mm Hg—50 (40-60)52 (42-60) Cardiac index, L/min/m2—2.09 (1.76-2.43)2.07 (1.70-2.36) PVR, dynes/sec/cm5—821 (506-1197)842 (596-1177)NE, ng/mL97.6 (74.4-126.6)205.1 (123.6-387.3)169.9 (119.1-310.3)Elafin, ng/mL45.5 (28.1-92.8)32.0 (15.3-59.1)42.4 (33.1-72.2)NE to elafin ratio2.0 (0.8-3.8)6.2 (3.0-17.3)4.3 (2.0-7.2)Data are presented as No. (%) or median (interquartile range). Missing data: all PAH group, 6MWD (n = 5), Dlco (n = 20), RAP (n = 4), CI and PVR (n = 2); follow-up group: Dlco (n = 10). 6MWD = 6-min walk distance; APAH = associated pulmonary arterial hypertension; CHD = congenital heart disease; CTD = connective tissue disease; D&T = drugs and toxins; Dlco = diffusion capacity of lung for carbon monoxide; — = data not available for healthy control participants; ERA = endothelin receptor antagonist; HPAH = heritable pulmonary arterial hypertension; IPAH = idiopathic pulmonary arterial hypertension; mPAP = mean pulmonary arterial pressure; NE = neutrophil elastase; NT-proBNP = N-terminal B-type natriuretic peptide; NYHA FC = New York Heart Association functional class; PAH = pulmonary arterial hypertension; PDE-5 = phosphodiesterase-5; PoPH = portopulmonary hypertension; PVR = pulmonary vascular resistance.a HPAH: n = 3 patients (1.2% of cohort), who had confirmed BMPR2 mutations. Mutation status was not otherwise evaluated routinely in the cohort. Open table in a new tab Data are presented as No. (%) or median (interquartile range). Missing data: all PAH group, 6MWD (n = 5), Dlco (n = 20), RAP (n = 4), CI and PVR (n = 2); follow-up group: Dlco (n = 10). 6MWD = 6-min walk distance; APAH = associated pulmonary arterial hypertension; CHD = congenital heart disease; CTD = connective tissue disease; D&T = drugs and toxins; Dlco = diffusion capacity of lung for carbon monoxide; — = data not available for healthy control participants; ERA = endothelin receptor antagonist; HPAH = heritable pulmonary arterial hypertension; IPAH = idiopathic pulmonary arterial hypertension; mPAP = mean pulmonary arterial pressure; NE = neutrophil elastase; NT-proBNP = N-terminal B-type natriuretic peptide; NYHA FC = New York Heart Association functional class; PAH = pulmonary arterial hypertension; PDE-5 = phosphodiesterase-5; PoPH = portopulmonary hypertension; PVR = pulmonary vascular resistance. Relative to control participants, patients with PAH were observed to have increased plasma NE levels (median, 205.1 ng/mL [IQR, 123.6-387.3 ng/mL] vs 97.6 ng/mL [IQR, 74.4-126.6 ng/mL]), decreased elafin levels (median, 32.0 ng/mL [IQR, 15.3-59.1 ng/mL] vs 45.5 ng/mL [IQR, 28.1-92.8 ng/mL]), and higher NE to elafin ratio (median, 6.2 [IQR, 3.0-17.3] vs 2.0 [IQR, 0.8-3.8]; all P < .0001) (Fig 1A, 1B ). The magnitude of NE level elevation was similar across PAH subtypes. Elafin was deficient for all subtypes except portopulmonary hypertension. The NE level better discriminated patients with PAH than elafin (c-statistic, 0.811 [CI, 0.767-0.853] vs 0.650 [CI, 0.590-0.710]) (Fig 1C), and ideal cutoffs were NE level of > 134.8 ng/mL (specificity, 0.83; sensitivity, 0.70) and elafin level of < 35.9 ng/mL (specificity, 0.65; sensitivity, 0.56). The NE to elafin ratio did not improve PAH discriminatory power (c-statistic, 0.808 [CI, 0.763-0.852]), and no positive or negative correlation existed between NE and elafin levels in patients with PAH or healthy participants (e-Fig 2A, 2B). NE and elafin levels were independent of age in patients with PAH and healthy participants (e-Fig 3A). No sex-related NE differences were observed, although female patients with PAH exhibited more pronounced elafin deficiency (e-Fig 3B). Neither biomarker was related to race or ethnicity or to BMI (e-Fig 3C, 3D). Clinical markers of disease severity were associated with NE levels, but not elafin levels. The NE level increased with more severe heart failure symptoms from New York Heart Association functional classes I to IV (Fig 2A). Higher NE levels also correlated with shorter 6-min walk distance (ρ = –0.30; P = .006), increased N-terminal pro-type brain natriuretic peptide (ρ = 0.25; P = .011), worse right ventricular function (tricuspid annular plane systolic excursion ρ = –0.23; P = .019), and higher mPAP (ρ = 0.21; P = .039) (e-Table 2). Relationships persisted after adjustment for age, sex, and BMI, where each two-fold NE elevation equated to a 6-min walk distance reduction of 34.2 m, relative N-terminal pro-type brain natriuretic peptide increase of 20.2%, tricuspid annular plane systolic excursion reduction of 0.18 cm, and mPAP increase of 4.1 mm Hg (Fig 2B). NE and elafin levels were independent of time from PAH diagnosis (e-Fig 4), PAH treatment status (naïve, monotherapy, dual therapy, or triple therapy) (e-Fig 5), each class of background PAH therapy (phosphodiesterase-5 inhibitors, endothelin receptor antagonists, or prostanoids) (e-Fig 6), and background immune modulators (e-Fig 7). Median follow-up for outcomes was 3.4 years (IQR, 2.0-5.8 years), during which time 52 patients (20.9%) died and 19 patients (7.6%) underwent transplantation. The 5-year survival rate was more favorable in the lowest NE quartile (85.3% [CI, 76.2%-95.5%]) than higher quartiles (quar
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