Background Several studies have identified circulating biomarkers to be associated with the presence and severity of pulmonary arterial hypertension (PAH). Recent evidence supports a role for galectin-3 (Gal-3) and the mineralcorticoid aldosterone in left ventricular failure. However, studies on aldosterone together with Gal-3 in PAH are lacking.
Objective We investigated a novel Aldosterone-galectin-3 (Gal-3) tandem and several other potential PAH biomarkers and their association with the disease severity.
Methods A total of 57 patients, 41 with idiopathic PAH. (IPAH) and 16 with PAH associated with connective tissue disease (CTD), and 8 age-matched, non-relative controls were studied. Gal-3, aldosterone and other potential protein plasma concentrations were measured by single ELISA and multi-array MSD (Meso Scale Discovery) technology.
Results Gal-3 values were increased in both patients with IPAH (12.2±0.6 ng/mL; p<0.05) and with PAH-CTD (14.1±1.6 ng/mL; p<0.05) versus control (8.5±0.9 ng/mL), while aldosterone was significantly elevated in IPAH only (248.5±38.8 pg/mL vs control 71.9±18.2 pg/mL; p<0.05). In addition, aldosterone, Gal-3, and N-terminal pro-brain natriuretic peptide (NT-proBNP) values were all higher in patients in WHO functional class II–III versus PAH functional class I or controls. The vascular injury marker intercellular adhesion molecule 1 (ICAM-1) was increased in IPAH and PAH-CTD versus controls (559.5±18.2 pg/mL and 734.1±59.4 pg/mL vs controls 394.8±39.3 pg/mL, p<0.05, p<0.0001, respectively), whereas vascular cell adhesion molecule 1 (VCAM-1) and proinflammatory, anti-angiogenic interleukin-12 (IL-12) were elevated in PAH-CTD only (879.5±110.0 pg/mL and 391.2±70.3 pg/mL vs controls 489.8±44.6 pg/mL, p<0.01, and 102.1±15.2 pg/mL, p<0.01, respectively).
Conclusions Heightened Gal-3 and aldosterone plasma concentrations in PAH patients indicate a role for Gal-3 signalling in the pathobiology of IPAH and PAH-CTD, and may serve as biomarkers for functional status and progression of disease.
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Pulmonary arterial hypertension (PAH) and associated heart failure is a complex, multifactorial disease with a poor prognosis.1–3 None of the current therapies has been shown to be universally effective, nor have these therapies been able to reverse advanced pulmonary vascular disease or to prevent right heart failure in advanced PAH.2 ,3 The progressive nature and heterogeneous aetiology of PAH underlines the critical need for both early diagnosis and accurate stratification to allow tailored, efficient therapies.4
Several studies have identified circulating biomarkers associated with PAH, including markers of endothelial dysfunction, vascular injury and remodelling, myocardial damage, inflammation, and oxidative stress. N-terminal pro-brain natriuretic peptide (NT-proBNP), endothelin-1, interleukin-6, interferon-α, C reactive protein (CRP), serotonin, as well as endothelial progenitor cells, and recently identified pulmonary hypertension (PH)-relevant microRNAs, belong to the most validated markers.5 ,6
A role for aldosterone in pulmonary vascular remodelling and fibrosis has been suggested by increased plasma and tissue concentrations of aldosterone in experimental models, and also in patients with PAH.7–10 Aldosterone contributes to the activation of pathways that decrease bioavailable nitric oxide levels, induce inflammation, and promote cell proliferation, migration, extracellular matrix remodelling, and fibrosis. Moreover, clinical mineralocorticoid receptor (MR) blockade in addition to endothelin-1 receptor inhibition has been associated with a better prognosis in clinical PAH.11
The molecular mechanisms by which aldosterone exerts its cardiovascular effects are complex.12 Recent evidence indicates a critical role for galectin-3 (Gal-3), a β-galactoside-binding lectin, in inflammation, fibrosis, and heart failure, downstream of aldosterone.13 ,14 Accordingly, Gal-3 is already established as a biomarker for cardiac fibrosis, and chronic left ventricular heart failure.15 ,16 Consequently, Gal-3 has been approved by the US Food and Drug Administration as a new biomarker for additive risk stratification in heart failure, and received a class IIb recommendation in the most recent American Heart Association/American College of Cardiology (AHA/ACC) guidelines.17
However, to date, combined clinical studies on the role of both Gal-3 and aldosterone in PAH are lacking. Here, we investigate several biomarkers in PAH associated with vascular injury and myocardial fibrosis, and identify Gal-3 and aldosterone as potential tandem biomarkers in patients with idiopathic PAH (IPAH) or PAH associated with connective tissue disease (PAH-CTD).
During the research conferences of the Pulmonary Hypertension Association in California (2010) and Florida (2012), 57 patients (IPAH, n=41; PAH-CTD, n=16) and eight healthy, age- and gender-matched, non-relative controls were recruited. Exclusion criteria were male sex, sleep apnoea, liver disease, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis. All participants completed a questionnaire regarding their demographic data (tables 1 and 2) and medication (table 2). Written informed consent was obtained from each subject. The study has been approved by the internal review board of Northeastern University (IRB # 11-07-19). Detailed characteristics of each individual PAH patient can be found in table 2.
EDTA whole blood samples were collected through peripheral venipuncture in non-fasting subjects, processed to plasma (ie, immediate centrifugation for 10 min at 1300 g), and stored at −80°C. Gal-3 and aldosterone concentrations were measured in one thaw step by BGM Gal-3 ELISA (BG Medicine, Waltham, Massachusetts, USA) and by aldosterone ELISA (RE52301, IBL International, Hamburg, Germany), respectively, according to the manufacturers’ instructions. For assessment of NT-proBNP, the singleplex sandwich electrochemiluminescent immunoassay by Meso Scale Discovery was used, according to the manufacturer's protocol. All other molecules were measured using Meso Scale Discovery's Multi-Array technology (MSD, Angiogenesis Panel 1 Human, Cytokine Panel 1 Human, Proinflammatory Panel 1 Human, Vascular Injury Panel 2 Human). Signal intensities were measured with the MESO QuickPlex SQ 120 instrument (Meso Scale Discovery, Rockville, Maryland, USA) and analysis was performed using the Discovery Workbench software V.4.0 (Meso Scale Discovery, Rockville, Maryland, USA), according to the manufacturer's instructions. All protein markers of which more than 50% of the measurements fell into the detection range of the standard curves were considered valid.
Statistical analyses were performed with GraphPad Prism V.6 software. Data are expressed as mean±SEM or median values with IQR as appropriate. Data were tested for normal distribution with D'Agostino-Pearson omnibus, Shapiro-Wilk, and Kolmogorov-Smirnov tests. For comparison of data, one-way analysis of variance (ANOVA) or Kruskal-Wallis tests were used, corrected for multiple comparisons by Dunn's test, as appropriate. Values of p<0.05 were considered significant.
Demographic characteristics of the study subjects by groups are listed in table 1. Detailed information on each individual PAH patient including age, weight, body mass index, PAH diagnosis (IPAH, PAH-CTD), WHO functional class (FC) and medication is provided in table 2.
Plasma concentrations of Gal-3, aldosterone and NT-proBNP in PAH patients
In order to test whether Gal-3, aldosterone and NT-proBNP plasma concentrations were increased in patients with PAH versus controls, ELISAs were performed in control (n=8), IPAH (n=41), and CTD (n=16) plasma. Aldosterone plasma concentrations were elevated only in IPAH (248.5±38.8 pg/mL vs control 71.9±18.2 pg/mL; p<0.05), while the difference between controls and PAH-CTD patients (147.8±28.7 pg/mL vs control 71.9±18.2 pg/mL) did not reach statistical significance (figure 1A, B). Gal-3 concentrations were significantly increased in patients with IPAH (12.2±0.6 ng/mL) and PAH-CTD (14.1±1.6 ng/mL) versus controls (8.5±0.9 ng/mL; p<0.05; figure 1C, D). In addition, NT-proBNP values were increased in both IPAH and PAH-CTD patients as compared to controls (1575±291 ng/mL; p<0.05; and 1544±380 ng/mL; p<0.05, respectively; figure 1E, F).
Subsequently, we evaluated whether plasma concentrations of aldosterone, Gal-3, and NT-proBNP differed between PAH patients, grouped by WHO FC (FC I, n=10; FC II–III, n=53). Only one patient fulfilled the criteria for WHO FC IV and was, therefore, excluded from the analysis. Aldosterone, Gal-3, and NT-proBNP concentrations were all significantly increased in FC II-III PAH versus controls. A significant difference between FC II-III and FC I PAH was found only for Gal-3 and NT-proBNP but not for aldosterone plasma concentrations (figure 2A-B, C-D, E-F, respectively).
Other markers of vascular injury and inflammation
For our analysis we used several panels of the Meso Scale Discovery's Multi-Array assays in control (n=5), IPAH (n=41), and CTD (n=16) plasma. Quantitative measurements were considered reliable when more than 50% of the measurements fell within the linear range of the respective standard curves. Such markers included intercellular adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule 1 (VCAM-1), serum amyloid A (SAA), CRP, interleukin-12 (IL-12), fibroblast growth factor 2 (FGF-2) (figure 3), as well as interleukin-16 (IL-16), vascular endothelial growth factor (VEGF), VEGF-C, and VEGF-D (figure 4). Plasma ICAM-1 concentrations were increased in patients with IPAH and PAH-CTD versus controls (559.5±18.2 pg/mL and 734.1±59.4 pg/mL vs controls 394.8±39.3 pg/mL, p<0.05, p<0.0001, respectively). Of note, the average plasma ICAM-1 concentration was significantly higher in PAH-CTD than in IPAH patients (p<0.01; figure 3A). In addition, VCAM-1 and IL-12 values were elevated in PAH-CTD patients versus controls (879.5±110.0 pg/mL and 391.2±70.3 pg/mL vs controls 489.8±44.6 pg/mL, p<0.01, and 102.1±15.2 pg/mL, p<0.01, respectively; figure 3B, E), but not in IPAH patients. The vascular injury/inflammatory markers SAA and CRP plasma concentrations tended to be higher in IPAH and PAH-CTD versus controls (ns; figure 3C, D). FGF-2, that has previously been shown to be involved in the pathobiology of PAH,18 also showed a trend toward increased plasma concentrations in IPAH patients versus controls (ns; figure 3F). For the other protein biomarkers measured in PAH and control plasma (IL-16, VEGF, VEGF-C, VEGF-D), no statistically significant differences or clear trends were found (figure 4).
Biomarkers hold the potential to provide valuable insights into the pathobiology and disease progression of PAH—a heterogeneous, fatal disease leading to pressure overload and ultimately failure of the right ventricle. Prior biomarker studies identified several plasma proteins being altered in PAH.19 However, none of these molecules exhibited all characteristics of an ideal biomarker (eg, high specificity and sensitivity, correlation with disease progression and therapy, etc). Thus, a multiparameter approach has been proposed to adequately reflect the complex and diverse pathogenetic mechanisms of clinical PAH.6
This is the first report to show that both plasma aldosterone and Gal-3 are increased in PAH, associated with WHO functional class, thus pointing to the potential of aldosterone/Gal-3 to indicate disease severity. Gal-3 is involved in several pathological and physiological processes—for example, the regulation of organ development, immune reactions, tumorigenesis, tumour growth, and metastasis.20 In the heart, Gal-3 augments fibrosis and modulates immune responses, which confers to maladaptive cardiac remodelling.21 Extracellular matrix remodelling is a key component of fibrosis, and increased Gal-3, which is released by macrophages, is likely to reflect profibrotic activity.21 Since advanced pulmonary hypertensive vascular disease is characterised by the development of vascular and cardiac fibrosis, increased Gal-3 concentrations in PAH may reflect vascular and right ventricular fibrosis.22
Gal-3 is activated by aldosterone in vascular smooth muscle cells of the systemic circulation.13 Here, we demonstrate that aldosterone is significantly increased in IPAH, but not in PAH-CTD patients, indicating the involvement of aldosterone-independent pathways in CTD, for example, inflammation-mediated induction of Gal-3, possibly mediated by VCAM-1 and IL-12 (figure 2).20 Independently of the aldosterone pathway, Gal-3 is expressed by activated macrophages and endothelial cells, attracting inflammatory cells and inducing fibrosis.20 This aldosterone-independent Gal-3 secretion might be particularly active in PAH-CTD patients. Interestingly, we did not find a direct correlation between plasma Gal-3 and aldosterone in IPAH patients. Therefore, one might speculate that aldosterone-independent pathways are also relevant in IPAH-associated Gal-3 upregulation. However, we provide data from a single time point, so that further studies addressing the temporal courses of aldosterone and Gal-3 values, and their possible correlation, are needed. Further limitations of this study include uncontrolled dietary and volume status at the time of plasma acquisition.
Both aldosterone and Gal-3 correlated with advanced disease states (as indicated by WHO-FC). It is therefore likely that longitudinal assessments of aldosterone and Gal-3 may help identify patients with more advanced PAH. Aldosterone can be targeted by well-established MR antagonists such as spironolactone, which raises the possibility of treating selected patients with aldosterone antagonists.10 ,23 In fact, cardiac remodelling and dysfunction appears to be responsive to MR blockade (spironolactone) treatment in clinical studies, such as the ARIES and TOPCAT trials.11 ,24
Because plasma aldosterone is not increased in PAH-CTD, while Gal-3 upregulation is observed in both IPAH and PAH-CTD, PAH patients might benefit from direct Gal-3 blockade. These issues therefore need to be addressed in future studies.
Beyond the aldosterone/Gal-3 tandem, we found that ICAM-1 was elevated both in IPAH and PAH-CTD versus controls. Interestingly, in our study, increased IL-12 and VCAM-1 concentrations were only found in PAH-CTD but not in IPAH patients, which indicates PAH-subtype specific roles for these molecules, probably related to the degree of inflammation, vascular injury and anti-angiogenesis. In serum from PAH patients, IL-12 expression values were found to be moderately elevated in IPAH and strongly elevated in PAH-CTD.25 In contrast, patients with chronic thromboembolic pulmonary hypertension, who do not show perivascular infiltration of T and B lymphocytes in the lungs, had no significant changes in the circulating concentrations of IL-12, indicating that IL-12 elevation is not a direct response to elevated pulmonary arterial pressure.25 The association of circulating VCAM-1 and ICAM-1 with PH was also reported in patients with sickle cell disease.26 Knockdown of BMPR-II increased ICAM-1 and VCAM-1 expression in HUVECs (human umbilical vein endothelial cells).27 These data suggest that VCAM-1 and ICAM-1 induction reflects endothelial activation and dysfunction occurring in PAH.
Taken together, we provide evidence for an involvement of Gal-3 and aldosterone in the pathobiology of human PAH and right ventricular dysfunction, and suggest their potential application as a new tandem in clinical PAH. This study provides the rationale for further clinical and experimental studies investigating the aldosterone/Gal-3 axis in the pathogenesis of PAH. Larger prospective studies including hemodynamics are needed to assess the suitability of aldosterone/Gal-3 as tandem biomarkers for disease progression and treatment response in different PAH aetiologies.
What is already known on this subject?
The mineralocorticoid receptor agonist aldosterone is involved in pulmonary hypertensive vascular disease and cardiovascular fibrosis. We have previously demonstrated in animal models and in vitro that galectin-3 (Gal-3) is a key mediator of cardiac and renal fibrosis induced by aldosterone.
What might this study add?
This study demonstrates for the first time that the axis aldosterone/galectin-3 (Gal-3) is relevant in pulmonary arterial hypertension (PAH), by showing that plasma levels of both are associated with PAH severity.
How might this impact on clinical practice?
Galectin-3 (Gal-3) is already established as a biomarker for cardiac fibrosis, and chronic left heart failure. This work suggest potential applications for aldosterone/galectin-3 (Gal-3) as a new tandem biomarker for clinical PAH.
We thank Dr Paolo Galuppo for his advice on the aldosterone ELISA.
LC, EL and LG contributed equally.
Contributors GH conceived the study, and obtained IRB approval, samples and written consent. AH, BDP, HS and SKM participated in the study design, IRB approval and sample collection. LC, EL, and LG performed the biochemical assays and statistical analysis. LG, HS, and GH structured and analysed the clinical data sets. GH and HS wrote the paper. HS, LC, EL, JB and GH edited the manuscript.
Funding GH currently receives grant support from the German Research Foundation (DFG; HA 4348/2-1), Kinderherzen e.V (W-H-001-2014), and Stiftung KinderHerz (2511-6-13). LC is the recipient of a postdoctoral research stipend from the European Section of the Aldosterone Council (ESAC). SKM was supported by grant R01 EB009327 from the US National Institutes of Health. The contribution of the COST Action ADMIRE BM1301 is acknowledged (JB).
Competing interests None declared.
Patient consent Obtained.
Ethics approval Northeastern University, Boston (IRB # 11-07-19).
Provenance and peer review Not commissioned; externally peer reviewed.