Metabolomics reveals metabolite changes of patients with pulmonary arterial hypertension in China

Abstract The specific mechanism of pulmonary arterial hypertension (PAH) remains elusive. The present study aimed to explore the underlying mechanism of PAH through the identity of novel biomarkers for PAH using metabolomics approach. Serum samples from 40 patients with idiopathic PAH (IPAH), 20 patients with congenital heart disease‐associated PAH (CHD‐PAH) and 20 healthy controls were collected and analysed by ultra‐high‐performance liquid chromatography coupled with high‐resolution mass spectrometry (UPLC‐HRMS). Orthogonal partial least square‐discriminate analysis (OPLS‐DA) was applied to screen potential biomarkers. These results were validated in monocrotaline (MCT)‐induced PAH rat model. The OPLS‐DA model was successful in screening distinct metabolite signatures which distinguished IPAH and CHD‐PAH patients from healthy controls, respectively (26 and 15 metabolites). Unbiased analysis from OPLS‐DA identified 31 metabolites from PAH patients which were differentially regulated compared to the healthy controls. Our analysis showed dysregulation of the different metabolic pathways, including lipid metabolism, glucose metabolism, amino acid metabolism and phospholipid metabolism pathways in PAH patients compared to their healthy counterpart. Among these metabolites from dysregulated metabolic pathways, a panel of metabolites from lipid metabolism and fatty acid oxidation (lysophosphatidylcholine, phosphatidylcholine, perillic acid, palmitoleic acid, N‐acetylcholine‐d‐sphingomyelin, oleic acid, palmitic acid and 2‐Octenoylcarnitine metabolites) were found to have a close association with PAH. The results from the analysis of both real‐time quantitative PCR and Western blot showed that expression of LDHA, CD36, FASN, PDK1 GLUT1 and CPT‐1 in right heart/lung were significantly up‐regulated in MCT group than the control group.


| INTRODUC TI ON
Pulmonary arterial hypertension (PAH) is a chronic disease condition involving in vascular remodelling disease of the lungs, which causes an increase in the pulmonary artery pressure and eventually leading to right heart failure and death. Despite the progress in knowledge has been obtained about potential therapeutic targets and introduction of newer drugs, the 5-year survival rate still remains low. 1 This can be attributed to the ill-understood pathophysiology of PAH, necessitating further exploration of the mechanism of disease development for the development of novel therapeutic strategies.
In recent years, the major efforts have been focused on the differential regulation in the metabolic pathways which may be contributory to PAH pathogenesis. Emerging evidence points towards a metabolic theory of PAH, suggesting that PAH results from the suppression of the mitochondria-based respiration and glucose oxidation (named Warburg effect in cancer metabolism). 2,3 It causes cells to rapidly proliferate without undergoing apoptosis of cells and accelerates the vascular remodelling in PAH. It is widely regarded that various metabolic changes during the Warburg effect are also essential for the occurrence and maintenance of PAH. 3,4 In addition, metabolic changes involving fatty acid oxidation and amino acid breakdown are also thought to be involved in the formation of PAH. 5 A deeper understanding of these pathways holds the potential to provide targets for the diagnosis and treatment of PAH. However, due to the complex nature of these metabolic pathways in PAH, a single metabolic pathway affecting the disease pathogenesis and progression is difficult to illustrate. However, large scale metabolomics provides a lucrative alternative to understand the differential regulation of these metabolic pathways involved in PAH. In a seminal study by Zhao et al, 6  tricarboxylic acid intermediates and purine metabolites. 7 Reports from recent study are also congruent with these findings where they demonstrate a strong strength of association between patient survival and metabolic profiles in PAH. 8 However, the metabolic profile of individuals is dependent on race, sex and dietary habits. Till now, no studies focusing on the metabolomic changes in a primarily Chinese PAH patient cohort have been documented. The current study aimed to fill this gap in knowledge in our study. Employing ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UPLC-HRMS) in a non-targeted metabolomics analysis, we aim to explore the metabolic profiles in plasma from PAH patients (idiopathic or congenital heart disease (CHD) associated PAH) and their healthy counterparts. We also investigated the differentially expressed targets in PAH rat model to delineate the changes in the key enzymes involved in glucose and lipid metabolism. This is intended to identify the signature of metabolites for PAH, which can be used for biomarker discovery and potential therapeutic targets for the disease germane. After fasting for 8 hours before right-side heart catheterization, venous blood samples were drawn from the femoral venous and collected in EDTA anticoagulant tubes, centrifuged (1000 g, 10 minutes) and stored at −80°C until required.

| Sample preparation and UPLC-HRMS
The method of sample preparation for the metabolomic analysis was in accordance with the sample preparation method by UPLC-HRMS. 11 Briefly, serum samples were prepared by 10 µL serum Nitrogen was used as both cone gas (50 L/h) and desolvation gas (600 L/h). Source temperature and desolvation temperature were set at 120°C and 350°C, respectively. The capillary voltage and cone voltage were 3000 and 20 V, respectively. Chlorpropamide (5 µmol/L) was added in the sample as the internal standard. A volume of 10 µL sample from each plasma ample was prepared as a quality control (QC) sample to validate the stability of sequence analysis. The QC sample was extracted and analysed in the same way as describe above. In order to evaluate the repeatability, QC sample and blank (pure acetonitrile) sample were injected after every 10 samples during the analytic run.

| Measurement of RVSP and RVH
Haemodynamic measurement was performed as previously described. 14 Rats were anaesthetized by intraperitoneal injection of pentobarbital sodium. A venous catheter was inserted in the right jugular vein and introduced in the right atrium (RA) of rats to observe the RVSP. After haemodynamic measurement, the lungs and hearts were harvested. The RV and left ventricular (LV) plus interventricular septum (S) were separated and then weighed, respectively. Right ventricular hypertrophy (RVH) was using the ratio of RV weight to LV plus S weight [RV/(LV + S)].

| Total RNA preparation and real-time quantitative PCR analysis
Quantitative real-time PCR was performed as previously described. 15 Total RNA was extracted from lungs or heart tissue using TRIzol (Invitrogen) according to the manufacturer's instructions.
The RNA was reverse-transcribed to cDNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific) and was performed with gene-specific primers and SYBR Green Master Mix (Applied Biosystems). The data were normalized to GAPDH. The transcripts were assessed by real-time PCR on a 7300 qPCR system (all from Applied Biosystems). Relative gene expression was quantified using the comparative threshold cycle value (∆CT) method with the above primers. The relative gene expression was calculated as fold change = 2 −∆(∆Ct) .

| Western blotting analysis
Western blotting analysis was performed as previously described. 16 Briefly, the proteins from harvested lung were extracted with RIPA lysis buffer (Beyotime) and separated on sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) before transferring onto polyvinylidene difluoride (PVDF) membranes. Subsequently, the membranes were blocked with 5% milk PBST solution and then incubated with the following respective antibodies: rabbit monoclonal anti-CD36, rabbit monoclonal anti-GLUT1, rabbit mono-

| Statistical analysis
Comparisons between multiple groups were conducted using oneway analysis of variance (ANOVA) followed by least significant difference post hoc tests. Comparisons between two groups were conducted using Student's t test. The difference with P < .05 (two sides) was statistically significant. Potential biochemical markers were further evaluated by receiver operating characteristic (ROC) analysis. The area under the ROC curve (AUC) is to assess the sensitivity and specificity of the biomarkers. AUC values >0.9 indicate high reliability of the model, 0.7-0.9 indicate moderate reliability, 0.5-0.7 indicate poor reliability and AUC ≤ 0.5 suggests that the model prediction is not better than chance.

| Clinical characteristics
The levels of NT-proBNP, uric acid and total bilirubin in IPAH patients were significantly higher than normal and only the levels of total bilirubin in CHD-PAH patients were significantly higher than normal value ( Table 2). The results of cardiac colour ultrasound showed the diameter of right ventricle (RV) and right atrium in IPAH patients was larger than normal value. The diameter of RV in CHD-PAH patients was larger than normal value ( Table 2). The results of right-side heart catheterization showed RVSP, RVDP, mPVP and pulmonary vessel resistance were significantly higher than normal value both in IPAH and CHD-PAH patients ( Table 2).

| Metabolomic profiles
The results of PCA showed there were no remarkable outliers, and the stability of the method was high (data not shown). Mass Profnder and Mass Profler Professional software were used to extract the raw data and conducted peak extraction, the peak cluster as well as retention time correction. Ultimately, we got the data about massto-charge ratio (m/z), the retention time (RT) and the data matrix of  (Table S1).
And S-plot model was applied to figure out the differential variable. When VIP value of the metabolite was greater than 1.0, it is considered as a significant contribution to the model. Student's t test was used to analyse the difference between two groups.  (Table S2). Among these metabolites, 31 metabolites from PAH patients were significantly up-regulated (n = 22) or down-regulated (n = 9) compared with respective metabolites from healthy controls (Table S2, Figure 2A,B).

| Enrichment and clustering of metabolites of interest
The metabolic pathway enrichment and clustering analysis showed

| ROC curve analysis of metabolites
In order to assess the diagnostic capacity of the metabolites, the receiver operating characteristic (ROC) curve analysis was applied to the data sheet. The area under the ROC curve for LysoPC, PC, decanoylcarnitine and l-carnitine was 97%, 83%, 79% and 74.4% in IPAH (n = 40)

| Haemodynamic index of MCT-induced PAH rats
In the second week (day 14) and the third week (day 21), mean pulmonary arterial pressure (mPAP) ( Figure S1A Figure S1D).

| Changes of genes and proteins involved in glucose and fatty acid metabolism in lungs of MCTinduced PAH rat model
We

| Changes of genes and proteins involved in glucose and fatty acid metabolism in right hearts of MCT-induced PAH rat model
We

| D ISCUSS I ON
Our study provides insight into the metabolic signatures of PAH with the potential to unravel novel biomarkers and therapeutic targets. In in myocardium and accounts for approximately 70% of fatty acid uptake into contracting cardiomyocytes. 28 Our study found that CD36 expression was significantly up-regulated in lungs and hearts of MCT-induced PAH, which was consistent with previous studies in the field. 29  induce pulmonary artery smooth muscle proliferation, vasoconstriction and microthrombosis and play a key role in the development of PAH. 32 Therefore, the decrease in tryptophan in our study may be related to the increased conversion of tryptophan to serotonin, which is contributory to the pulmonary artery endothelial proliferation and vascular remodelling. In addition, we found increased levels of l-phenylalanine in patients with IPAH, compared with healthy controls. This is significant considering the fact that ratio of (phenylalanine/tyrosine) elevation is considered as a biochemical marker for endothelial dysfunction, 33 emphasizing the endothelial dysfunction in our cohort of PAH patients.
In present study, we found an increase in levels of phosphatidylcholine (PC) and a decrease in levels of lysophosphatidylcholine (LysPC) in the PAH group, compared with that in the healthy controls. However, their role in disease pathogenesis is unclear and is an area of active research.

F I G U R E 6
Changes of genes and proteins involved in glucose and fatty acid metabolism in the hearts of the MCT-induced PAH rat model. Male SD rats (180 g) randomly received an intraperitoneal injection of normal saline (control, n = 12) or monocrotaline (MCT, n = 24) to induce PAH. The rats in control group were examined at the third week (day 21), and rats in MCT group were randomly examined at the second (day 14, MCT-2 wk, n = 12) and third week (day 21, MCT-3 wk, n = 12). The mRNA expressions of LDHA (a), CD36 (b), CPT-1 (c), FASN (d) and PDK4 (e) were tested by real-time PCR (A), relative protein levels of proteins were tested by Western blot (B), relative levels of LDHA, CD36, cpt-1β and FASN were calculated (C). *P < .05; **P < .01 In conclusion, metabolic profiling in a Chinese population indicates towards a metabolic pathogenesis of PAH, highlighting the significance of these metabolites in biomarker discovery and devising therapeutic strategies.

CO N FLI C T O F I NTE R E S T
The authors have no conflict of interest to disclose.

DATA AVA I L A B I L I T Y S TAT E M E N T
Raw data will be made available on request.