Targeted proteomics of right heart adaptation to pulmonary arterial hypertension

Abstract

No prior proteomic screening study has centred on the right ventricle (RV) in pulmonary arterial hypertension (PAH). This study investigates the circulating proteomic profile associated with right heart maladaptive phenotype (RHMP) in PAH.Plasma proteomic profiling was performed using multiplex immunoassay in 121 (discovery cohort) and 76 (validation cohort) PAH patients. The association between proteomic markers and RHMP, defined by the Mayo right heart score (combining RV strain, New York Heart Association (NYHA) class and N-terminal pro-brain natriuretic peptide (NT-proBNP)) and Stanford score (RV end-systolic remodelling index, NYHA class and NT-proBNP), was assessed by partial least squares regression. Biomarker expression was measured in RV samples from PAH patients and controls, and pulmonary artery banding (PAB) mice.High levels of hepatocyte growth factor (HGF), stem cell growth factor-β, nerve growth factor and stromal derived factor-1 were associated with worse Mayo and Stanford scores independently from pulmonary resistance or pressure in both cohorts (the validation cohort had more severe disease features: lower cardiac index and higher NT-proBNP). In both cohorts, HGF added value to the REVEAL score in the prediction of death, transplant or hospitalisation at 3 years. RV expression levels of HGF and its receptor c-Met were higher in end-stage PAH patients than controls, and in PAB mice than shams.High plasma HGF levels are associated with RHMP and predictive of 3-year clinical worsening. Both HGF and c-Met RV expression levels are increased in PAH. Assessing plasma HGF levels might identify patients at risk of heart failure who warrant closer follow-up and intensified therapy.

FIGURE 1

Study design. PAH: pulmonary artery hypertension; PLS: partial least squares; RHMP: right heart maladaptive phenotype; RV: right ventricle; IF: immunofluorescence; PAB: pulmonary arterial banding; RT-qPCR: reverse transcriptase quantitative PCR.

FIGURE 2

a) Variable importance in projection (VIP) scores and correlations for each cytokine with respect to right heart scores and metrics in both cohorts and b) levels of the four markers according to levels of right heart (RH) maladaptive phenotype in patients with pulmonary arterial hypertension (PAH) and controls. a) Using partial least squares regression, we assessed the correlations between plasma proteomic markers (using multiplex immunoassay) and markers of right heart maladaptive phenotype (Stanford RH score based on right ventricular (RV) end-systolic remodelling index (RVESRI), New York Heart Association functional class (NYHA FC) and N-terminal pro-brain natriuretic peptide (NT-proBNP) levels; Mayo RH score based on RV free-wall longitudinal strain (RVLS), NYHA FC and NT-proBNP levels; RVESRI; RVLS; NT-proBNP; NYHA FC; mean pulmonary artery pressure (mPAP) and pulmonary vascular resistance (PVR)) in 121 (discovery cohort, from 2008 to 2011) and 76 (validation cohort, from 2012 to 2014) PAH patients. Positive correlations are shown in red, negative correlations in blue. RVLS is presented in absolute value (lowest values indicate worst RV dysfunction). b) Analyte levels of the four strongest biomarkers associated with right heart maladaptive phenotype according to tertiles of the i) Mayo RH score or ii) Stanford RH score in patients with PAH and in the 88 healthy controls. Comparison between each right heart risk category to controls was performed using Dunn's and Kruskal–Wallis test (across-groups among patients with PAH) with post hoc pairwise comparisons adjusted for multiple testing (Benjamini–Hochberg). **: p<0.01; ***: p<0.001; ****: p<0.0001. The p-values of the Cuzick test were used to confirm an increasing trend of cytokine levels across ordered categories (from low to intermediate to high). SCGF: stem cell growth factor; HGF: hepatocyte growth factor; NGF: nerve growth factor; SDF: stromal cell-derived factor; MIF: macrophage migration inhibitory factor; IFN: interferon; IL: interleukin; MCSF: macrophage colony-stimulating factor; VEGF: vascular endothelial growth factor; TNF: tumour necrosis factor; LIF: leukaemia inhibitory factor; GCSF: granulocyte colony-stimulating factor; GMCSF: granulocyte–macrophage colony-stimulating factor; TRAIL: TNF-related apoptosis-inducing ligand; FGF: fibroblast growth factor; PDGF: platelet-derived growth factor; SCF: stem cell factor.

FIGURE 3

Prognostic value of hepatocyte growth factor (HGF) and its incremental value to risk scores for outcome prediction. 5-year Kaplan–Meier survival curves for a) the primary combined end-point of death, lung transplant or hospitalisation for acute right heart failure of the total pulmonary arterial hypertension (PAH) cohort (n=197) and b) according to quartiles of HGF levels at baseline. c) Chi-squared of scores and HGF, nerve growth factor (NGF), stem cell growth factor (SCGF)β and stromal cell-derived factor (SDF)1 for prediction of the primary end-point (death, transplant or admission for right heart failure) at 3 years in the total PAH cohort (n=197) using Cox regression. Variables were entered in the model using enter mode. The Mayo right heart score was based on the New York Heart Association (NYHA) functional class, N-terminal pro-brain natriuretic peptide (NT-proBNP) and right ventricular longitudinal strain (RVLS). The Stanford right heart score was based on the NYHA class, NT-proBNP and right ventricular end-systolic remodelling index (RVESRI). The REVEAL score was based on the Registry to Evaluate Early and Long-term PAH Disease Management (REVEAL) groups: low, average, moderate high, high and very high. *: p<0.05; **: p<0.01.

FIGURE 4

Immunoblot of the protein expression of hepatocyte growth factor (HGF), its receptor c-Met, nerve growth factor (NGF) and its receptor TrkA, stem cell growth factor (SCGF)β and and stromal cell-derived factor (SDF)1 in the right ventricle of patients with pulmonary arterial hypertension (PAH) and controls (CTRL), and quantification of the signal. Each bar represents the mean±sem signal intensity of Western blots lysate from right ventricular samples from four PAH patients undergoing heart–lung transplant and five control patients (heart donors), which were compared using t-tests. GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

FIGURE 5

Localisation of a) c-Met (red) and b) its ligand hepatocyte growth factor (HGF; red) in right ventricular biopsies from a patient with idiopathic pulmonary arterial hypertension by immunostaining, double-labelled in green with i) TNNT2 troponin (cardiomyocyte), ii) α-smooth muscle actin (SMA), iii) CD31 (endothelial cells), iv) vimentin (VMTN) (fibroblast) or v) CD68 (macrophage). No immunoreactivity was seen in cells incubated with the secondary antibody but no primary antibody. Scale bars=10 μm. DAPI: 4′,6′-diamidino-2-phenylindole.