Abstract
Cytokines can modulate vascular remodelling and the adaptation of the right ventricle in pre-capillary pulmonary hypertension (PH). However, detailed data on the circulating levels of cytokines in patients are limited. We measured blood cytokine concentration in 39 treatment-naïve patients (pulmonary arterial hypertension: N = 16, chronic thromboembolic PH: N = 15, PH due to lung disease: N = 8) and 12 control subjects using enzyme-linked immunoassays. Apelin concentration >1,261 ng/mL identified patients with PH (66% sensitivity and 82% specificity), and in patients it was related to systolic pulmonary arterial pressure (PAP) (r = 0.33, P = 0.04), right atrial pressure (r = 0.38, P = 0.02), cardiac index (r = −0.34, P = 0.04), and right ventricular stroke work index (r = −0.47, P = 0.003). IL22RA2 concentration correlated with mean PAP (r = −0.32, P = 0.04) and serum N-terminal pro B-type natriuretic peptide level (r = −0.42, P = 0.01). VEGF concentration increased in patients upon clinical improvement (N = 16, P = 0.02). Circulating apelin is a novel biomarker of pre-capillary PH. Apelin and IL22RA2 levels are related to right ventricular function upon diagnosis of PH.
Introduction
Pre-capillary pulmonary hypertension (PH) is a rare disease, which often progresses and can impose a high risk of mortality [1]. Patients with pre-capillary PH include those with pulmonary arterial hypertension (PAH), PH associated with lung diseases, and chronic thromboembolic PH (CTEPH) or PH with unclear and/or multifactorial mechanisms. PAH and CTEPH are characterised by pulmonary vascular remodelling, which is induced by the interplay between mechanical stress, cellular hypoxia, and metabolic changes in the endothelium [2]. As PH progresses and the right ventricular pressure load continuously increases, adaptive and later maladaptive right ventricular remodelling processes occur, and poor disease prognosis is associated with right ventricular dysfunction and failure [3]. Myocardial remodelling is a complex process involving the apoptosis of cardiomyocytes, neurohormonal activation, and changes in cellular metabolism due to ischemia [2]. Non-invasive biomarkers, which are associated with disease severity, progression, and therapeutic response, could aid diagnosis and follow-up and improve patient outcomes [4].
Angiogenic and inflammatory pathways have been described to promote the formation of pulmonary vascular lesions, and cytokine effects are also linked with right ventricular function [2, 5]. Vascular endothelial growth factor (VEGF) induces the proliferation of vascular endothelial and smooth muscle cells in PAH [6, 7]; however, VEGF could also play a role in adaptive right heart remodelling [2] and an increased circulating VEGF level is associated with better right ventricular function in patients [8].
Similarly, apelin also regulates the survival of vascular endothelial cells [9], controls the proliferation of pulmonary artery smooth muscle cells [10], and the loss of apelin signalling is associated with a more severe PH in an animal model due to the downregulation of endothelial nitric oxide (NO) synthase in pulmonary arteries [11]. Importantly, the infusion of apelin-13 decreased pulmonary vascular resistance and increased cardiac output in patients with PAH [12], but data on circulating apelin levels in patients with PH are limited and contradictory. Goetze et al. reported a decrease in plasma apelin-36 concentration in patients with idiopathic PAH [13]. In line with this, Chandra and colleagues found lower serum apelin-12 concentrations in patients with PAH than in controls [11]. However, Foris and colleagues reported an increased concentration of circulating apelin-17 in patients with idiopathic PAH (IPAH) [14].
In addition, interleukin-22 receptor A2 (IL22RA2), a soluble receptor blocking IL-22 effects [15], has recently been suggested to be linked to the preservation of right heart function in an animal model of PH, and an increased circulating IL22RA2 concentration was noted in untreated patients with IPAH [16].
Importantly, blood cytokine quantification can aid the diagnosis and prognostic evaluation of patients with PAH [14, 17, 18]. However, detailed human data on the association of VEGF, apelin and IL22RA2 with clinical parameters in pre-capillary PH are lacking.
Therefore, we hypothesized that elevated levels of circulating VEGF, apelin, and IL22RA2 could serve as diagnostic biomarkers in patients with PAH and CTEPH, and that the changes in these parameters are linked with improved cardiac function during patient follow-up. Hence, firstly, we compared mediator levels in untreated patients and control subjects and among subgroups of PH, and studied the correlation between cytokine levels and functional, haemodynamic, and echocardiographic parameters in patients. We also assessed the value of these cytokines in detecting pre-capillary PH. Furthermore, we studied the dynamics of cytokine concentrations after treatment in patients with IPAH and CTEPH upon clinical improvement.
Materials and methods
Subjects and study design
39 patients with pre-capillary PH and 12 control volunteers were enrolled into the study at the Cardiopulmonary Unit, Department of Pulmonology, Semmelweis University, Budapest, Hungary, between June 2017 and September 2020. During the diagnostic work-up, patients underwent detailed examinations including the collection of previous medical data, echocardiography, pulmonary angio-CT, lung perfusion scans (for suspicion of pulmonary thromboembolism), right heart catheterization (RHC), lung function test, blood tests, 6-min walk test and the New York Heart Association (NYHA) Functional Class (FC) was determined [1]. Pre-capillary PH was established according to current guidelines [1]: mean pulmonary artery pressure (mPAP) > 20 mmHg, pulmonary vascular resistance (PVR) > 2 Wood units (WU), pulmonary capillary wedge pressure (PCWP) ≤ 15 mmHg.
In addition, venous blood samples were stored for cytokine measurements. Lung diseases were diagnosed by a pulmonary specialist of our Interstitial Lung Disease (ILD) Team when necessary. 16 patients were diagnosed with PAH (IPAH n = 13, PH associated to portal hypertension N = 2, PH due to connective tissue disease N = 1), 15 patients had CTEPH, and 8 patients had PH associated with lung diseases (ILD N = 5, chronic obstructive pulmonary disease N = 3). In 16 patients (PAH N = 12, CTEPH N = 4), echocardiography, 6-min walk test, and blood tests were repeated, and venous blood samples were stored during control visits after therapy. Control subjects were volunteers working at the Department, who did not have signs of heart failure and did not have any uncontrolled chronic medical condition. Medical history was taken, echocardiography, lung function tests were performed, and blood samples were stored for later analysis.
The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Ethics Committee of Semmelweis University, Budapest, Hungary (protocol code 91/2017; date of approval: 26 June 2017). All subjects provided a written informed consent.
Measurements of clinical parameters
RHC: The investigation was performed with the patient in a supine position, and the pressure transducer was set to zero level at the mid-thoracic point. Routine parameters were registered including right atrial pressure (RAP), right ventricular pressure, PAP, and PCWP. Cardiac output (CO) was measured using thermodilution. During the catheterisation of IPAH patients, vasoreactivity tests were performed with inhaled iloprost for 15 min. Furthermore, the stroke volume (SV = CO/heartbeat), SV index (SVI = SV/body surface area), PVR=(mean PAP-PCWP)/CO, and cardiac index (CI = CO/body surface area) were calculated [1, 19]. Additional parameters were determined to assess the severity of pulmonary arterial remodelling and systolic right ventricle function, including pulmonary arterial compliance (PAC = SV/(systolic PAP – diastolic PAP)) [20], and right ventricular stroke work index (RVSWI= (mean PAP – RAP) x SVI x 0.0136) [21].
Echocardiography
Echocardiography was performed using a Mindray DC-70 X-Insight instrument (Shenzhen Mindray Bio-Medical Electronics Co., Shenzen, China) to determine specific right heart parameters, such as right atrial area, right ventricular diameter, tricuspid annular plane systolic excursion (TAPSE), right ventricular outflow tract velocity time integral (RVOT VTI), right ventricular outflow tract acceleration time (RVOT AT) and the estimated right ventricular systolic pressure [1, 22].
Blood tests: N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentration and arterial blood gases were measured in patients.
Lung function tests: All subjects underwent spirometry, body plethysmography, and at the same time, lung diffusing capacity was measured [23–25].
6-min walk testing: 6MWT was performed in a standard 20-m-long corridor with continuous monitoring of oxygen saturation and heart rate. Before and after the test, blood pressure and Borg score were determined [26].
Enzyme-linked immune-assay (ELISA) measurements
Serum and EDTA plasma were collected from the subjects, spun down at 3,000 rpm for 10 min, and stored at −80 °C for later analysis. Serum samples were used for the quantification of VEGF-A [8] and apelin, while IL22R2A concentration was measured in plasma [16] using commercially available assays according to the manufacturers' instructions (VEGF-A: Human VEGF ELISA kit, Proteintech Europe, Manchester, UK, detection limit: 6.5 pg/mL; apelin (detecting all active forms including apelin-36, apelin-31, apelin-28 and apelin-13): RayBio Human/Mouse/Rat Apelin C-Terminus Enzyme Immunoassay Kit, RayBiotech, Norcross, GA, USA, detection limit: 15.8 ng/mL; IL22RA2: RayBio Human IL-22BP ELISA Kit, RayBiotech, Norcross, GA, USA, detection limit: 1.64 ng/mL). Apelin was measured in 63 samples (patients with PH upon enrolment N = 38, control visit N = 14; control subjects N = 11) and VEGF and IL22RA2 concentrations were quantified in 67 samples (patients with PH upon enrolment N = 39, control visit N = 16; control subjects N = 12). Half of the detection limit value, i.e. 3.25 pg/mL, was assigned to samples with a VEGF concentration below the detection limit. In the samples taken upon enrolment, 17 patients had VEGF concentration below the detection limit (IPAH: N = 6, CTEPH: N = 7, PH due to lung disease: N = 4), and among them, 2 patients with IPAH and 2 patients with CTEPH also had VEGF concentrations below the detection limit in samples collected during the follow-up visit (altogether 21 samples from patients with VEGF concentration below the detection limit). In addition, 6 samples from controls also showed VEGF level below the detection limit. Apelin and IL22RA2 concentrations were above the detection limit in all samples.
Statistical analysis
Data were analysed using t-test and ANOVA with Bonferroni post hoc analysis, and data are expressed as mean ± standard deviation. Cytokine concentrations did not show a normal distribution (either before or after log transformation), therefore Mann-Whitney test, Wilcoxon signed rank test, Kruskal-Wallis test with Dunn's post hoc analysis and Spearman correlation were used to assess the relationship between cytokine concentrations and clinical factors listed in Table 1. Data are expressed as median (interquartile range). P < 0.05 was considered significant. For data analysis and electronic artwork creation, the GraphPad Prism 9.1 software package was used (GraphPad Software, San Diego, USA). The ability of apelin to identify patients with PH was assessed with the receiver operating characteristic (ROC) curve and optimum cut-off values were chosen using the Youden index (MedCalc version 19., MedCalc software Ltd, Ostend, Belgium).
Subject characteristics
| Variables | Control | All PH | P-value | PAH | CTEPH | PH due to lung diseases | P-value |
| Number | 12 | 39 | NA | 16 | 15 | 8 | NA |
| Sex, male/female N | 6 (50%)/6 (50%) | 18 (46%)/21 (54%) | 0.99 | 6 (38%/10 (72%) | 7 (47%)/8 (53%) | 4 (50%)/4 (50%) | 0.81 |
| Age, years | 57 ± 10 | 59 ± 13 | 0.68 | 59 ± 12 | 58 ± 14 | 61 ± 14 | 0.83 |
| BMI, kg/m2 | 27.6 ± 4.6 | 29.1 ± 5.8 | 0.63 | 29.5 ± 6.0 | 29.2 ± 6.0 | 28.2 ± 5.9 | 0.87 |
| Non-smoker/ex-/Smoker, N | 7/3/2 (58%/25%/17%) | 28/11/0 (72%/28%/0%) | 0.48 | 11/5/0 (69%/31%/0%) | 12/3/0 (80%/20%/0%) | 5/3/0 (63%/37%/0%) | 0.63 |
| Pack-years | 17 ± 14 | 25 ± 15 | 0.37 | 25 ± 17 | 14 ± 6 | 40 ± 15 | 0.21 |
| Haemodynamic variables: | |||||||
| sPAP, mmHg | NA | 75 ± 15 | NA | 76 ± 14 | 73 ± 17 | 79 ± 13 | 0.41 |
| mPAP, mmHg | NA | 48 ± 9 | NA | 49 ± 8 | 47 ± 12 | 50 ± 6 | 0.82 |
| dPAP, mmHg | NA | 32 ± 9 | NA | 34 ± 8 | 21 ± 10 | 33 ± 8 | 0.44 |
| RAP, mmHg | NA | 15 ± 8 | NA | 14 ± 6 | 15 ± 6 | 15 ± 7 | 0.20 |
| PCWP, mmHg | NA | 11 ± 3 | NA | 11 ± 3 | 11 ± 3 | 11 ± 3 | 0.99 |
| SVI, mL/m2/beat | NA | 26.7 ± 7.7 | NA | 26.3 ± 7.3 | 27.0 ± 6.4 | 27.4 ± 11.2 | 0.95 |
| CI, L/min/m2 | NA | 2.1 ± 0.6 | NA | 2.0 ± 0.5 | 2.3 ± 0.7 | 2.2 ± 0.6 | 0.31 |
| PVR, Wood Unit | NA | 10.5 ± 4.7 | NA | 10.8 ± 4.3 | 8.5 ± 4.5 | 13.3 ± 5.5 | 0.07 |
| PAC, mL/mmHg | NA | 1.26 (0.83–1.62) | NA | 1.23 (0.84–2.01) | 1.27 (0.94–1.56) | 1.25 (0.72–1.36) | 0.79 |
| RVSWI, g/m2/beat | NA | 12.40 ± 5.05 | NA | 12.43 ± 4.34 | 12.02 ± 5.72 | 12.99 ± 5.74 | 0.91 |
| Echocardiographic variables: | |||||||
| sPAP, mmHg | 28 ± 6 | 72 ± 18 | <0.001 | 73 ± 15 | 67 ± 21 | 80 ± 19 | 0.28 |
| RV, mm | 27 ± 2 | 40 ± 7 | <0.001 | 42 ± 7 | 39 ± 7 | 38 ± 5 | 0.36 |
| TAPSE, mm | 27 ± 5 | 17 ± 5 | <0.001 | 16 ± 5 | 18 ± 5 | 18 ± 3 | 0.33 |
| RA area, cm2 | 11.6 ± 2.8 | 25.0 ± 10.2 | <0.01 | 25.5 ± 8.6 | 26.5 ± 12.6 | 20.8 ± 7.7 | 0.47 |
| RVOT VTI, cm | 13.9 ± 1.0 | 10.4 ± 3.7 | <0.05 | 9.7 ± 3.7 | 12.2 ± 3.6 | 8.6 ± 2.7 | 0.08 |
| RVOT AT, ms | 178 ± 48 | 72 ± 26 | <0.001 | 74 ± 25 | 72 ± 26 | 68 ± 30 | 0.88 |
| Lung function variables: | |||||||
| FVC %ref | 105 ± 12 | 87 ± 24 | 0.07 | 94 ± 19 | 95 ± 24 | 66 ± 18**## | <0.01 |
| FEV1 %ref | 100 ± 9 | 83 ± 24 | 0.07 | 89 ± 15 | 91 ± 28 | 57 ± 15**## | <0.01 |
| FEV1/FVC | 0.79 ± 0.05 | 0.76 ± 0.10 | 0.32 | 0.76 ± 0.09 | 0.74 ± 0.05 | 0.74 ± 0.16 | 0.22 |
| RV %ref | 135 ± 40 | 119 ± 50 | 0.41 | 118 ± 34 | 119 ± 52 | 118 ± 75 | 0.99 |
| TLC %ref | 115 ± 18 | 97 ± 22 | 0.05 | 100 ± 20 | 100 ± 15 | 81 ± 35 | 0.06 |
| RV/TLC | 0.39 ± 0.05 | 0.44 ± 0.15 | 0.36 | 0.43 ± 0.11 | 0.41 ± 0.17 | 0.54 ± 0.16 | 0.15 |
| KCO %ref | NA | 54 ± 23 | NA | 50 ± 25 | 66 ± 14 | 39 ± 20# | <0.05 |
| DLCO %ref | NA | 64 ± 27 | NA | 60 ± 29 | 82 ± 15* | 38 ± 12*### | <0.001 |
| Blood tests: | |||||||
| pH | NA | 7.42 ± 0.04 | NA | 7.42 ± 0.04 | 7.42 ± 0.04 | 7.41 ± 0.04 | 0.70 |
| PaO2, mmHg | NA | 61.1 ± 13.3 | NA | 60.6 ± 13.9 | 60.3 ± 12.3 | 52.2 ± 9.6# | <0.05 |
| PaCO2, mmHg | NA | 33.1 ± 6.8 | NA | 30.9 ± 5.1 | 32.7 ± 6.9 | 38.6 ± 7.2* | <0.05 |
| NT-proBNP, pg mL | NA | 1,489 (600–2,778) | NA | 1,489 (608–2,642) | 1,665 (505–2,887) | 972 (439–3,393) | 0.89 |
| Functional categories and tests: | |||||||
| NYHA I, N | NA | 0 (0%) | NA | 0 (0%) | 0 (0%) | 0 (0%) | 0.12 |
| NYHA II, N | NA | 10 (26%) | NA | 3 (19%) | 7 (47%) | 0 (0%) | |
| NYHA III, N | NA | 28 (72%) | NA | 13 (81%) | 7 (47%) | 8 (100%) | |
| NYHA, IV, N | NA | 1 (2%) | NA | 0 (0%) | 1 (6%) | 0 (0%) | |
| 6MWD, m | NA | 339 ± 140 | NA | 317 ± 142 | 400 ± 124 | 261 ± 130 | 0.06 |
| Circulating cytokine concentrations: | |||||||
| Serum apelin, ng/mL | 952 (782–1,261) | 1,434 (1,070–1,636) | <0.05 | 1,510 (1,115–1,750) | 1,313 (936–1,493) | 1,448 (985–1,753) | 0.60 |
| Serum VEGF, pg/mL | 5.78 (3.25–12.89) | 7.39 (3.25–10.26) | 0.96 | 7.11 (3.25–9.18) | 7.44 (3.25–10.86) | 5.32 (3.25–21.00) | 0.99 |
| Plasma IL-22RA2, ng/mL | 201 (145–668.6) | 236.5 (88.7–523.3) | 0.83 | 200.6 (99.8–427.6) | 172.8 (34.7–677.8) | 328.3 (204.5–535.9) | 0.68 |
Data are presented as mean ± standard deviation or median (interquartile range). Patients and controls are compared with the Fisher exact test, t-test or Mann-Whitney test. Patient subgroups are analysed using the chi-square test, ANOVA with Bonferroni post-hoc test or Kruskal-Wallis test with Dunn's post-hoc test. *P < 0.05, **P < 0.01 vs. PAH; #P < 0.05, ##P < 0.01, ###P < 0.001 vs. CTEPH.
Abbreviations: BMI: body mass index, CI: cardiac index, CTEPH: chronic thromboembolic PH, DLCO: diffusing capacity of the lung for carbon monoxide, FEV1: forced expiratory volume in the 1st second, FVC: forced vital capacity, IL-22RA2: interleukin-22RA2, KCO: carbon monoxide transfer coefficient for, N: number, NA: not applicable, NT-proBNP: N-terminal pro-B-type natriuretic peptide, NYHA FC: New York Heart Association Functional Class, PaO2/CO2: arterial partial pressure of O2/CO2, PAH: pulmonary arterial hypertension, PAC: pulmonary artery compliance, PH: pulmonary hypertension, s/m/dPAP: systolic/mean/diastolic pulmonary artery pressure, RAP: right atrial pressure, PVR: pulmonary vascular resistance, PCPW: pulmonary capillary wedge pressure, RA: right atrium, ref: reference, RV: right ventricle, RV: residual volume, RVOT AT/VTI: right ventricular outflow tract acceleration time/velocity time integral, RVSWI: right ventricular stroke work index, SVI: stroke volume index, TAPSE: tricuspid annular plane systolic excursion, TLC: total lung capacity, VEGF: vascular endothelial growth factor, 6MWD: six-minute walk distance.
Results
Subject characteristics
There were no differences in age, sex, body mass index, smoking habits, or lung function parameters between patients with PH and control subjects (Table 1). RHC results demonstrated moderate to severe increases in mean PAP and PVR with a normal or decreased cardiac index in patients. Vasoreactivity was not observed in any of the patients with IPAH. A mild hypoxaemia with hypo- or normocapnia was noted in the patients, which was most prominent in the group of patients with PH associated with lung disease. NT-proBNP levels were increased, and most patients fell into NYHA functional classes III and IV. Haemodynamic and echocardiographic variables and NT-proBNP concentrations were not different among the subgroups of pre-capillary PH.
Comparison of circulating cytokine concentrations in patients and controls
Serum apelin concentrations were higher in patients with PH than in control subjects (Table 1). Using the cut-off value of >1,261 ng/mL apelin concentration, patients could be identified with a sensitivity of 66% and a specificity of 82% (ROC area under the curve (AUC) = 0.74, P = 0.001). Nonetheless, our data did not show a difference in the circulating concentrations of IL22RA2 and VEGF between patients and controls (Table 1), and these mediators could not separate patients from controls (ROC AUC = 0.52, P = 0.96 and ROC AUC = 0.52, P = 0.83, respectively).
Cytokine concentrations were not different among PH subgroups (Table 1). In patients, VEGF concentration showed a moderate positive correlation with both apelin (r = 0.33, P = 0.04) and IL22RA2 levels (r = 0.42, P = 0.008), but no association was found between IL22RA2 and apelin concentrations (r = 0.14, P = 0.41).
The relationship between cytokine concentration and clinical factors in untreated PH
Serum apelin concentration was positively correlated with systolic PAP (r = 0.33, P = 0.04, Fig. 1A) and right atrial pressure (r = 0.38, P = 0.02), but it showed a negative relationship with SVI (r = −0.58, P < 0.001), cardiac index (r = −0.34, P = 0.04, Fig. 1B), PAC (r = −0.55, P < 0.001, Fig. 1C) and RVSWI (r = −0.47, P = 0.003, Fig. 1D). Plasma IL22RA2 concentrations showed a negative correlation with mean PAP (r = −0.32, P = 0.04, Fig. 2A) and serum NT-proBNP level (r = −0.42, P = 0.01, Fig. 2B), but not with other clinical factors (P > 0.05). VEGF concentration was not related to any of the clinical parameters (P > 0.05).
Spearman correlation between serum apelin concentrations and systolic pulmonary arterial pressure (sPAP – panel A), cardiac index (panel B), pulmonary artery compliance (PAC – panel C) and right ventricular stroke work index (RVSWI) (panel D) in patients with PH
Citation: Physiology International 2023; 10.1556/2060.2023.00264
Spearman correlation between serum apelin concentrations and systolic pulmonary arterial pressure (sPAP – panel A), cardiac index (panel B), pulmonary artery compliance (PAC – panel C) and right ventricular stroke work index (RVSWI) (panel D) in patients with PH
Citation: Physiology International 2023; 10.1556/2060.2023.00264
Spearman correlation between serum apelin concentrations and systolic pulmonary arterial pressure (sPAP – panel A), cardiac index (panel B), pulmonary artery compliance (PAC – panel C) and right ventricular stroke work index (RVSWI) (panel D) in patients with PH
Citation: Physiology International 2023; 10.1556/2060.2023.00264
Spearman correlation between plasma IL22RA2 concentrations and mean pulmonary artery pressure (mPAP - panel A) and N-terminal pro-brain natriuretic peptide (NT-proBNP – panel B) in patients with PH
Citation: Physiology International 2023; 10.1556/2060.2023.00264
Spearman correlation between plasma IL22RA2 concentrations and mean pulmonary artery pressure (mPAP - panel A) and N-terminal pro-brain natriuretic peptide (NT-proBNP – panel B) in patients with PH
Citation: Physiology International 2023; 10.1556/2060.2023.00264
Spearman correlation between plasma IL22RA2 concentrations and mean pulmonary artery pressure (mPAP - panel A) and N-terminal pro-brain natriuretic peptide (NT-proBNP – panel B) in patients with PH
Citation: Physiology International 2023; 10.1556/2060.2023.00264
Circulating cytokine concentrations in PAH and CTEPH before and after treatment
We also compared cytokine concentrations before and after therapy in 16 patients. 12 patients with IPAH had a second visit after the initiation of pulmonary vasodilator therapy (phosphodiesterase 5 inhibitor N = 7, phosphodiesterase 5 inhibitor plus endothelin receptor antagonist N = 5). Furthermore, in 4 patients with CTEPH, data collection was repeated after successful pulmonary endarterectomy, and one patient required additional therapy with a phosphodiesterase 5 inhibitor. The time difference between the two measurements was 7 ± 3 months in IPAH and 15 ± 7 months in CTEPH. There was an improvement in NYHA functional status after therapy, and we found a tendency for an increase in TAPSE, RVOT VTI and 6MWD (Table 2).
Clinical variables in patients with IPAH or CTEPH before and after therapy
| Variables | Before treatment N = 16 | After treatment N = 16 | P-value |
| Serum apelin, ng/mL N=14 | 1,412 (1,165–1,619) | 1,343 (1,085–1,552) | 0.95 |
| Serum VEGF, pg/mL N=16 | 7.76 (3.25–8.49) | 9.37 (4.16–12.22) | 0.001 |
| Plasma IL-22RA2, ng/mL N=16 | 225.2 (143–574.1) | 320.7 (109.1–578.4) | 0.43 |
| 6MWD, m N=11 | 372 ± 143 | 435 ± 142 | 0.06 |
| NYHA FC N=16 | 3 (3–3) | 1 (1–3) | <0.001 |
| NT-proBNP, pg/mL N=14 | 1,412 (586–2,158) | 800 (183–1,832) | 0.30 |
| sPAP, mmHg N=16 | 69 ± 16 | 62 ± 18 | 0.21 |
| RV, mm N=16 | 40 ± 7 | 38 ± 7 | 0.33 |
| TAPSE, mm N=15 | 17 ± 5 | 19 ± 5 | 0.05 |
| RA area, cm2 N=15 | 27 ± 14 | 27 ± 11 | 0.95 |
| RVOT VTI, cm N=12 | 10.2 ± 3.6 | 11.7 ± 3.6 | 0.06 |
| RVOT AT, ms N=12 | 76 ± 30 | 84 ± 24 | 0.54 |
Data are presented as mean ± standard deviation or median (interquartile range). Data are compared with paired t-test or Wilcoxon signed rank test. Abbreviations: IL-22RA2: interleukin-22RA2, N: number, NA: not applicable, NT-proBNP: N-terminal pro-B-type natriuretic peptide, NYHA FC: New York Heart Association Functional Class, sPAP: systolic pulmonary artery pressure, right atrium, RV: right ventricle, RVOT AT/VTI: right ventricular outflow tract acceleration time/velocity time integral, TAPSE: tricuspid annular plane systolic excursion, VEGF: vascular endothelial growth factor, 6MWD: six-minute walk distance.
The concentration of circulating VEGF increased after treatment (P < 0.05), but the levels of apelin and IL22RA2 remained unchanged (Table 2). Interestingly, the change in VEGF concentration showed an inverse correlation with the change in RVOT VTI (r = −0.74, P < 0.01, Fig. 3), but not with the other parameters listed in Table 2 (P > 0.05).
Spearman correlation between the change in serum VEGF and the change in right ventricular outflow tract velocity time integral (RVOT VTI) after treatment
Citation: Physiology International 2023; 10.1556/2060.2023.00264
Spearman correlation between the change in serum VEGF and the change in right ventricular outflow tract velocity time integral (RVOT VTI) after treatment
Citation: Physiology International 2023; 10.1556/2060.2023.00264
Spearman correlation between the change in serum VEGF and the change in right ventricular outflow tract velocity time integral (RVOT VTI) after treatment
Citation: Physiology International 2023; 10.1556/2060.2023.00264
Discussion
Accumulating evidence shows that cytokines and inflammatory pathways contribute to the development of vascular remodelling, and they can also regulate right heart function in some forms of PH [2, 5, 14, 18], implicating that these mediators could serve as disease markers and future therapeutic targets. In the present study, we assessed the biomarker potential of three mediators in patients with pre-capillary PH. We showed that circulating apelin is increased in patients with pre-capillary PH, and it is associated with worse right heart function and vascular remodelling at diagnosis. Plasma IL22RA2 is related to the severity of heart failure on initial presentation of patients. In addition, serum VEGF levels are responsive to changes in the clinical condition of patients.
Apelin is a paracrine regulatory peptide expressed in many tissues including the systemic and pulmonary vasculature, heart, brain, adipose tissue, the endothelium of small pulmonary arteries, and the endo- and myocardium [9]. Relevant to PH, it signals via the APJ receptor found on human vascular smooth muscle cells, endothelial cells, and cardiomyocytes [27, 28]. Hypoxia, a major factor inducing pulmonary vasoconstriction, triggers apelin expression [29]. Signalling pathways known to be involved in the pathomechanism of PAH, such as bone morphogenetic protein receptor type 2 and hypoxia-induced factor-1, also regulate the expression of apelin and APJ [9, 30]. There is evidence that circulating apelin can be a marker of its cardiac expression. For example, apelin content in the right ventricle is increased in animals with chronic hypoxic PH, and it shows a positive correlation with right ventricular pressure and plasma apelin concentration [31]. Interestingly, apelin expression is increased in the left atrium and ventricle in patients with chronic left heart failure, and atrial apelin expression moderately correlates with plasma apelin concentrations, suggesting that it may be a source of circulating apelin [32]. To our knowledge, no study has explored the association between pulmonary/cardiac and circulatory apelin levels in patients with PH. However, it can be speculated that blood apelin concentration is related (at least to some extent) to cardiac apelin expression in PH.
Our study corroborates recent findings [14] that circulating apelin is increased in treatment-naïve patients with pre-capillary PH. We additionally showed that it is also a marker of cardiac function, as we found a link between mediator concentration and stroke volume, cardiac index, and right ventricular stroke work index. Previous data demonstrated that circulating apelin level seems to be specifically related to right ventricular failure, as it was not increased in patients with congestive heart failure caused by idiopathic dilated cardiomyopathy [33] or in patients with chronic heart failure due to coronary heart disease [32]. However, in an animal model of acute ischaemic left heart failure, the cardiac expression of both apelin and APJ receptors was elevated [34]. This suggests an initial upregulation of apelin signalling in early heart failure with a decrease in later stages [35], a mechanism also described in animal models of PH with adaptive and maladaptive right ventricular remodelling [30]. Thus, it can be hypothesized that in patients with pre-capillary PH, apelin production was induced in a compensatory manner to improve and maintain right heart function, as indicated by the increased mediator level at diagnosis and at the follow-up visit. This can also explain why other authors reported a decrease in circulating apelin concentration in patients with end-stage IPAH [13] or those already receiving therapies for PH [11].
Our data add to previous knowledge on the positive relationship between apelin production and cardiac output observed in a very small cohort of patients with PAH, where apelin expression in right ventricular tissue was positively related to cardiac output [30]. Furthermore, plasma apelin showed a positive relationship with mean PAP and right atrial pressure, which implies that pressure overload can induce apelin synthesis in cardiomyocytes and pulmonary endothelium. Interestingly, a higher apelin level was associated with reduced pulmonary vascular compliance, representing increased stiffness, pointing out a possible role of this mediator also in vascular remodelling, as it was described in relation with vessels of the systemic circulation [36].
In a recent study, Foris et al. reported the serum levels of different apelin isoforms in patients with PAH (apelin-12, apelin-13, apelin-17, apelin-36), and found that apelin-17 could distinguish patients from controls [14]. Here we report that the cumulative concentration of a partly similar spectrum of apelin isoforms (i.e. apelin-13, apelin-28, apelin-31, apelin-36) can also identify patients with pre-capillary PH, which does not contradict these previous findings as preproapelin is transformed via different routes into active apelin isoforms [37].
The involvement of IL22 in the pathogenesis of pulmonary hypertension has recently been described. IL22 induces cell growth of pulmonary artery smooth muscle via the activation of IL22 receptor alpha 1, and IL22 expression is upregulated in smooth muscle cells from rats with PH due to chronic hypoxia [38]. Moreover, using an in vivo model of chronic hypoxia-induced PH with right ventricular hypertrophy, IL22RA2, a decoy receptor for IL22 [15], was identified as a factor associated with adaptive right ventricular remodelling [16]. Our study adds to this observation as an increased circulating IL22RA2 concentration was related to lower NT-proBNP values. Furthermore, Crnkovic et al. reported an elevated concentration of plasma IL22RA2 in patients with untreated IPAH compared to controls [16]. However, in our cohort, we did not find a difference in mediator levels between patients and controls, which could partly be explained by the mixed population of patients with pre-capillary PH. In addition, IL22RA2 levels were not different among the PH subgroups, albeit the sample sizes of the subgroups were limited.
VEGF signalling has a complex effect during the development of vascular remodelling in response to different stimuli, as different receptor subtypes serve as positive and negative regulators of pulmonary endothelial cell proliferation [39]. Kümpers et al. demonstrated that the circulating concentration of VEGF was increased in patients with IPAH; however, they did not show an association with clinical parameters or disease progression [40]. We did not find a difference in plasma VEGF levels between patients with PH compared to controls, which can be due to the different disease aetiologies and the limited sample size in our cohort. In line with this, a recent study on a relatively large cohort of patients with untreated PAH did not a report a change in serum VEGF concentration between patients and matched controls [18]. Furthermore, VEGF signalling can also convert cardioprotective function, as its production is upregulated in the right ventricle during adaptive hypertrophy, and it can also trigger hypoxia-induced angiogenesis in the myocardium [5]. In line with this, we previously showed in a mixed population of treated and treatment-naïve PAH that TAPSE, a measure of right ventricular function, shows a positive relation to plasma VEGF concentration [8]. In the current study, we extend these findings by the observation that VEGF level is increased upon clinical improvement of patients after the initiation of specific treatment for PAH and CTEPH. However, the magnitude of the change in plasma VEGF level was inversely related to the improvement in RV outflow tract velocity time integral, which is a surrogate marker for right ventricular stroke volume reflecting both RV basal and free wall motion. This might be caused by a compensatory increase in right ventricular VEGF production to promote adaptive hypertrophy, as shown previously in an animal model of PH [41].
Interestingly, baseline VEGF concentration was positively associated with apelin and IL22RA2 levels. This can be explained by the fact that hypoxia can trigger the production of all three cytokines [29, 38, 41], and the expression of VEGF and IL22RA2 can also be induced by perivascular inflammation in remodelled pulmonary arteries [42].
Our study has limitations. The major limitation is the sample size of the patient groups. However, PAH and CTEPH are rare diseases, and PH is often undiagnosed in patients with lung diseases due to the current lack of evidence for the use of specific pulmonary vasodilator therapy in this group [1]. Future studies could extend our findings on the diagnostic potential of circulating apelin concentration by recruiting other control groups, including patients with lung disease, or those after pulmonary embolism without PH, or patients with less severe PH. Finally, similar to our previous study [8], despite using a detection assay with high sensitivity, VEGF concentration was below the detection limit in approximately one-third of the samples, which could have influenced the conclusions drawn from these data.
In conclusion, circulating apelin is a new biomarker of pre-capillary PH, with a possible link to right ventricular function. Our data provide further support for the association between IL22RA2 and VEGF signalling pathways and right heart function in PH. Future studies should explore the clinical benefits of measuring these circulating biomarkers during the initial and later clinical stages of pre-capillary PH.
Acknowledgements
We thank Tímea Baranyi for assistance in collecting clinical data. This work was supported by the Hungarian Respiratory Foundation to Dr. Györgyi Csósza [grant number MPA/2019].
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