Kynurenine Metabolites Predict Survival in Pulmonary Arterial Hypertension: A Role for IL-6/IL-6Rα

doi:10.21203/rs.3.rs-1222545/v1

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Kynurenine Metabolites Predict Survival in Pulmonary Arterial Hypertension: A Role for IL-6/IL-6Rα

https://doi.org/10.21203/rs.3.rs-1222545/v1

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Introduction: Activation of the kynurenine pathway (KP) has been reported in patients with pulmonary arterial hypertension (PAH) undergoing PAH therapy. We aimed to determine KP-metabolism in treatment-naïve PAH patients, investigate its prognostic values, evaluate the effect of PAH therapy on KP-metabolites and identify cytokines responsible for altered KP-metabolism.

Methods: KP-metabolite levels were determined in plasma from PAH patients (median follow-up 42 months) and in rats with monocrotaline- and Sugen/hypoxia-induced PH. Blood sampling of PAH patients was performed at the time of diagnosis, six months and one year after PAH therapy.

Results: KP activation with lower tryptophan, higher kynurenine (Kyn), 3-hydroxykynurenine (3-HK), quinolinic acid (QA), kynurenic acid (KA), and anthranilic acid was observed in treatment-naïve PAH patients compared with controls. A similar KP-metabolite profile was observed in monocrotaline, but not Sugen/hypoxia-induced PAH. Human lung primary cells (microvascular endothelial cells, pulmonary artery smooth muscle cells, and fibroblasts) were exposed to different cytokines in vitro. Following exposure to interleukin-6 (IL-6)/IL-6 receptor a (IL-6Ra) complex, all cell types exhibit a similar KP-metabolite profile as observed in PAH patients. PAH therapy partially normalized this profile in survivors after one year. Increased KP-metabolites correlated with higher pulmonary vascular resistance, shorter six-minute walking distance, and worse functional class. High levels of Kyn, 3-HK, QA, and KA measured at the latest time-point were associated with worse long-term survival.

Conclusion: KP-metabolism was activated in treatment-naïve PAH patients, likely mediated through IL-6/IL-6Ra signaling. KP-metabolites predict response to PAH therapy and survival of PAH patients.

pulmonary arterial hypertension

tryptophan

kynurenine pathway

inflammation

survival

Pulmonary arterial hypertension (PAH) is characterized by an increase in pulmonary vascular resistance due to pulmonary vascular remodeling. This increases right ventricular afterload, eventually leading to progressive right heart failure[1]. There is currently no therapy reversing pulmonary vascular remodeling. Hence early identification of the disease as well as delineation of novel mechanisms contributing to pulmonary vascular remodeling are still of utmost importance to improve prognosis of patients with PAH.

Recent studies have highlighted the pathophysiological importance of inflammation and mitochondrial dysfunction in the development and progression of PAH[2–4]. Nicotinamide adenine dinucleotide (NAD⁺) has been demonstrated to be an important modulator of inflammation and mitochondrial function[5–10]. NAD⁺ can be produced from vitamin B3 via the Preiss-Handler pathway and its derivate nicotinamide riboside via the salvage pathway[10]. Interestingly, NAD⁺ synthesis via the salvage pathway is enhanced in patients with advanced PAH as well as in rodent models of pulmonary hypertension (PH)[11]. However, a very important pathway that determines NAD⁺ levels is the de novo synthesis through the kynurenine pathway (KP). This de novo NAD⁺ synthesis through the KP starts with the conversion of essential amino acid tryptophan (Trp) into kynurenine (Kyn), by indoleamine 2, 3-dioxygenase, followed respectively by 3-hydroxykynurenine (3-HK), 3-hydroxykynurenic acid (3-HA), quinolinic acid (QA), and finally resulting in NAD⁺ formation[12, 13]. There are branching points in the KP, as Kyn is also metabolized to kynurenic acid (KA) and anthranilic acid (AA) (Figure 1)[12, 13]. Interestingly, a correlation between circulating Kyn, QA, and AA and pulmonary vascular resistance has been reported in PAH patients[14]. Similarly, increased Kyn levels were observed in other PAH cohorts[15, 16], and correlated with immune dysregulation and clinical outcome in a follow-up period of 6 months[16]. Together, these studies highlight the importance of the KP in PAH. However, PAH patients received PAH therapy in these studies and the KP-metabolite profile in treatment-naïve PAH patients as well as the effect of PAH therapy on this profile are currently unknown.

Therefore, we aimed to investigate: 1) KP-metabolite profile in treatment-naïve PAH patients as well as in two rat models of PAH, 2) the effects of PAH therapy on this profile in PAH patients, 3) the prognostic values of KP-metabolites during a long-term follow-up period. Given the potential correlation between KP-metabolism and immunity/ inflammation, we also investigated whether cytokines and hypoxia are able to change the KP-metabolism in human lung cells.

Study population

The study protocol was approved by the Erasmus MC ethical committee (MEC-2011-392 and 2012-512), and written informed consent was obtained from all PAH patients and healthy volunteers. All procedures were performed in accordance with Declaration of Helsinki. Patients were not involved in the design of the study.

In this prospective observational cohort study, which encompassed forty-three consecutive treatment-naïve adult patients with PAH (mean pulmonary artery pressure ≥ 25 mmHg and pulmonary artery wedge pressure ≤ 15 mmHg) diagnosed by right heart catheterization between May 2012 and October 2016 at Erasmus MC were included. Treatment-naïve was defined as the absence of any history of treatment with approved target medications for PAH, i.e. prostacyclin, endothelin receptor antagonists, or phosphodiesterase type 5 inhibitors, and the diagnosis of PAH was in accordance with definition at time of inclusion [17, 18]. Patients that aged <18 years, that were not treatment-naïve, had an incomplete diagnostic procedure, or were uncapable of signing informed consent were excluded. Thirty-nine PAH patients were prescribed PAH targeted therapies following diagnosis according to the latest guidelines[17, 19]. PAH patients were prospectively followed till the 1st of January 2019. The primary outcome was defined as all-cause mortality and lung transplantation. Survival status was checked in the Municipal Personal Records database.

A control group consisting of 111 healthy volunteers was selected from the cohort in the study of using 2D speckle tracking echocardiography to evaluate the normal myocardial strain values in healthy subjects. The selected subjects had normal results on physical examination and electrocardiography (ECG), had no (prior) cardiovascular disease or cardiovascular risk factors (hypercholesterolemia, hypertension (blood pressure above 140/90 mmHg at the time of visit), or diabetes mellitus). More details about these PAH and control cohorts have been previously described[20, 21].

Animal models of PH

Animal experiments were approved by the Ethics Committee of the Université Paris-Saclay (#11484) and carried out in accordance with the Guide for the Care and Use of Laboratory Animals adopted by the National Institute of Health and Medical Research (INSERM).and were performed in accordance with the ARRIVA guidelines. Plasma collected during previous experiments in two well-established rat models of severe PAH was used for this study[22]. Briefly, monocrotaline (MCT)-induced PH (MCT) was established in 4 weeks-old male Wistar rats (Janvier Labs, Saint Berthevin, France), with a single subcutaneous injection of MCT (40 mg/kg, Sigma-Aldrich, Saint-Quentin-Fallavier, France), and evaluated after 3 weeks (MCT-PH n=8, controls n=9). Sugen-hypoxia-induced PH (SuHx) was established in 4 weeks-old male Wistar rats (Janvier Labs, Saint Berthevin, France), by a single subcutaneous injection of SU5416 (20mg/kg, Sigma-Aldrich, Saint-Quentin-Fallavier, France) combined with exposure to normobaric hypoxia for 3 weeks followed by normoxia for 5 weeks (SuHx-PH n=10, controls n=5).

Human and rat EDTA-plasma samples

At the time of diagnosis (baseline), peripheral venous blood sampling was performed during diagnostic right heart catheterization for PAH patients, while blood sampling was performed at the time of visit for healthy volunteers. Blood sampling was also performed in surviving PAH patients 6 months (±3 months, n=32) and 1 year (±3 months, n=28) after PAH therapy. In the rats, blood sampling was performed under anesthesia (2% isoflurane) prior to excision of the heart and lungs.

In vitro study of cultured human primary lung cells

The effects of cytokines and hypoxia on KP-metabolism were studied in 3 different types of human primary lung cells from healthy donors: microvascular endothelial cells (MVECs, CC-2527, Lonza) from 2 donors, pulmonary artery smooth muscle cells (PASMCs, CC-2581, Lonza) from 2 donors, and lung fibroblast including human lung fibroblasts (CC-2512, Lonza) from 1 donor, and one MRC-5 lung fibroblast cell line (ATCC®CCL-171™). All cells were cultured in the corresponding medium kits. The passage of MVECs, PASMCs, and fibroblasts used in the final experiments were P8, P8, and P7, respectively. Hypoxia with 1% oxygen was achieved in a standard incubator with variable oxygen control (Thermo Fisher Scientific). The concentration of human recombinant cytokines (R&D systems) in the medium was 20 ng/mL for TNF-α (210-TA-020/CF), IL-6 (7270-IL-025/CF), IL-6/IL-6Rα (8954-SR/CF) and TGF-β1 (7754-BH-005/CF). MVECs were exposed to cytokines or hypoxia in basal medium with 0.5% FBS for 24 hours prior to collection of medium. PASMCs and fibroblasts were starved with serum-free medium for 24 hours and then exposed to cytokines or hypoxia in serum-free medium for 24 hours before collection of medium. The medium was centrifuged at 2,000 g for 20 minutes at 4°C and the clear supernatant was collected and stored in aliquots at −80°C.

Measurement of KP-metabolites

An in-house developed assay by ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was used to determine the KP-metabolite levels as previously described[23], using 10 µL sample mixed with 10 µL isotopically labeled internal standard, including deuterated Trp, Kyn, 3-HK, 3-HA, QA, KA, and AA (Buchem BV, Apeldoorn, The Netherlands).

Statistical analysis

Normality of continuous data was evaluated by Kolmogorov-Smirnov tests. Continuous variables are presented as mean±standard deviation (SD) or median [interquartile range (IQR)], categoric variables as numbers (percentages). Unpaired t-test or Mann-Whitney test were used to compare differences in continuous variables (e.g. metabolite levels) between 2 groups (e.g. human PAH vs controls). Wilcoxon matched-pairs signed rank test was used to compare differences in KP-metabolites between two time points (baseline vs 6 months, and baseline vs 1 year) in PAH patients. Chi-square test was used to compare the difference in categoric variables (e.g. sex, NYHA) between 2 groups (e.g. survivors vs non-survivors). Spearman correlation coefficients were used to determine correlations between different KP-metabolites, and correlations between KP-metabolites and baseline characteristics. Logistic regression was conducted to determine whether KP-metabolites were independent predictors that distinguishing PAH patients from healthy controls. Comparisons of survival between groups were performed using the Kaplan-Meier estimator with Breslow-Wilcoxon test and log-rank test. Univariable Cox proportional hazard regression were used to assess associations between KP-metabolite levels and mortality in PAH patients. Statistical analyses were performed using IBM SPSS software (version 21.0.0.1). P< 0.05 was considered statistically significant.

Characteristics of the study population

Baseline characteristics of treatment-naïve PAH patients at the time of diagnosis and healthy controls are summarized in Table 1. During a median follow-up of 42 [interquartile range: 32-58] months, twelve PAH patients (Non-survivors) reached the primary endpoint, the causes included end-stage heart failure (n=5), euthanasia because of end-stage cardiovascular and pulmonary disease (n=2), lung transplantation (n=1), progression of systemic sclerosis (n=1), multi-organ failure (n=1), sudden death presumed cerebral (n=1), and malignancy (n=1). Seven of them reached the endpoint within six months after diagnosis and hence had no follow-up measurements taken. Non-survivors were older, had higher heart rate and shorter 6-minute walking distance than survivors (Table 1).

Table 1

Baseline characteristics of all PAH patients and healthy controls
	Control	PAH
	Control	All	Survivors	Non-survivors
N	111	43	31	12
Aetiology
- iPAH, n (%) - CTD-PAH, n (%) - CHD-PAH, n (%)		15 (35) 17 (40) 11 (25)	14 (45) 8 (26) 9 (29)	1 (8) 9 (75) 2 (17)
Clinical characteristics
Age, years old	43±13	53±17 **	49±14	62±19 †
Sex, women n (%)	59 (53)	29 (67)	20 (65)	9 (75)
sBP, mmHg	123 [115-128]	122 [114-132]	123 [116-132]	115 [106-132]
HR, beats/min	68 [62-76]	78 [67-90] **	76 [63-87]	87 [76-99] †
BMI, kg/m²	23.8±2.9	27.0±6.1 ***	27.6±6.6	25.2±4.5
eGFR, mL/min/1.73m²	---	66.8±18.4	68.9±16.9	61.3±21.8
hs-CRP, mg/L	---	3.1 [1.5-10.5]	3.1 [1.2-9.0]	3.4 [2.1-22.3]
NYHA, I:II:III:IV	---	1:13:23:6	1:11:16:3	0:2:7:3
6MWD, m	---	337±153	377±136	198±133 ††
Right heart catheterization
mPAP, mmHg	---	50.5±16.1	50.5±15.9	50.6±17.3
PAWP, mmHg	---	11.8±5.6	11.6±6.0	12.2±4.9
PVR, WU	---	7.1 [5.1-11.8]	7.9 [5.6-12.0]	6.4 [4.2-11.3]
CO, L/min	---	4.7 [3.9-5.5]	4.6 [3.9-5.5]	4.9 [3.9-6.5]
CI, L/min/m²	---	2.5 [2.2-3.3]	2.5 [2.2-3.2]	2.5 [2.0-3.6]
PAH therapy types at time of censoring
- No therapy, n (%) - Mono-therapy, n (%) - Dual-therapy, n (%) - Triple-therapy, n (%)		4 (9.3) 9 (20.9) 13 (30.2) 17 (39.5)	2 (6.4) 4 (12.9) 11 (35.5) 14 (45.2)	2 (16.7) 5 (41.6) 2 (16.7) 3 (25.0)

Data are presented as mean ± SD, median [IQR], or numbers (percentages). **P<0.01, *** P <0.001 PAH versus control. †P<0.05, ††P<0.01, survivors versus non-survivors. Unpaired T Test, Mann-Whitney U Test, and Chi-Square. PAH: pulmonary arterial hypertension, iPAH: idiopathic PAH, CTD-PAH: connective tissue diseases associated PAH, CHD-PAH: congenital heart diseases associated PAH, sBP: systolic blood pressure, HR: heart rate, BMI: body mass index, eGFR: estimated glomerular filtration rate, hs-CRP: high-sensitivity C-reactive protein, NYHA: New York Heart Association classification, 6MWD: 6-minute walking distance, mPAP: mean pulmonary arterial pressure, PAWP: pulmonary arterial wedge pressure, PVR: pulmonary vascular resistance, CO: cardiac output, CI: cardiac index.

KP-metabolite profile in PAH

At the time of diagnosis (baseline), Trp was significantly lower in treatment-naïve PAH patients compared to controls, while Kyn, 3-HK, QA, KA and AA were significantly higher in treatment-naïve PAH patients and no significant difference in 3-HA was found (Figure 1B). Binary logistic regression analyses showed that KP-metabolites significantly distinguished PAH patients from controls at baseline both in a univariate model and when corrected for age, sex, and body mass index (Table 2). Moreover, manual stepwise logistic regression analyses showed that including the whole panel of altered metabolites in the model predicted better than only one metabolite by significantly increasing the Chi-square of the model (Table 2).

MCT-PH rats showed a similar KP-metabolite profile as that observed in PAH patients (Figure 1C), which was accompanied by an increase in NAD⁺ in the lungs from these rats (1.26 [0.81-1.46] in 6 healthy rats, vs 1.70 [1.43-1.92] µM/mg in 7 MCT-PH rats, P=0.035). Conversely, the KP-metabolite profile was different in SuHx-PH rats (Figure 1D). As the MCT-PH rats show the most severe inflammatory phenotype, these results suggest a link between inflammation and KP-metabolism in PAH.

Table 2

Prediction of PAH with each 1 µM decrease for Trp and 1 nM increase in other KP metabolites by binary logistic regression.
	Odds Ratio [95% CI]	P value	Chi-Square	P value
Univariable
Trp	1.047 [1.024-1.070]	<0.001	21.083	<0.001
Kyn	1.003 [1.002-1.004]	<0.001	39.370	<0.001
3-HK	1.097 [1.051-1.144]	<0.001	38.390	<0.001
QA	1.053 [1.032-1.075]	<0.001	56.114	<0.001
KA	1.076 [1.023-1.132]	0.005	8.842	0.003
AA	1.924 [1.453-2.550]	<0.001	40.132	<0.001
Whole panel			92.492	<0.001
Adjusted for age, sex and body mass index
Trp	1.036 [1.011-1.060]	<0.001	35.551	<0.001
Kyn	1.003 [1.002-1.005]	<0.001	57.983	<0.001
3-HK	1.089 [1.043-1.137]	<0.001	53.224	<0.001
QA	1.047 [1.026-1.069]	<0.001	63.944	<0.001
KA	1.090 [1.029-1.155]	0.003	35.368	<0.001
AA	1.870 [1.398-2.502]	<0.001	54.705	<0.001
Whole panel			93.187	<0.001

Trp: Tryptophan, Kyn: Kynurenine, 3-HK: 3-Hydroxykynurenine, QA: Quinolinic acid, KA: Kynurenic acid, AA: Anthranilic acid.

IL-6/IL-6Rα contributed to the activation of KP-metabolism in vitro

Stimulation with IL-6/IL-6Rα complex induced KP activation and mimicked the KP-metabolite profile observed in PAH patients in all three cell types, while stimulation with IL-6 alone failed to induce a similar profile (Figure 2A&B). Moreover, the cells responded differently to other cytokines and hypoxia failed to induce a KP profile similar to that seen in PAH patients (Figure 2C-E). Taken together, these results indicate that inflammation, particularly activation of IL-6/IL-6Rα signaling contributed to the KP activation in PAH patients.

Effects of PAH therapy on KP-metabolism

Following six months of PAH therapy, Trp was still significantly lower in PAH patients compared to controls, while only Kyn, 3-HK, QA, and AA were still significantly higher in PAH patients (Figure 1E-J). After one year of PAH therapy, Trp was still significantly lower in PAH patients than in controls, while only 3-HK and QA were still significantly higher in PAH patients (Figure 1E-J). These data suggest KP-metabolite profile partly normalized in PAH patients after PAH therapy.

When baseline KP-metabolite levels were compared between survivors and non-survivors, only Kyn was significantly higher in non-survivors versus survivors (Figure 3D). However, when comparing these levels at the latest measurement available, Kyn, 3-HK, QA, KA, and AA were all significantly higher in non-survivors compared with survivors (Figure 3, right panels). In these survivors, Wilcoxon matched-pairs signed rank test showed that Kyn, 3-HK, QA, KA, and AA were significantly decreased after one year but not six months of PAH therapy, indicating that only long-term PAH therapy decreased KP-metabolite levels (Figure 3, left panels).

These data indicate that KP-metabolites could be potential predictors of response to PAH therapy in survivors, and regular monitoring of KP-metabolites may be important to evaluate clinical status of PAH patients.

Correlation of KP-metabolites and disease-severity

Significant correlations between different KP-metabolites were seen in healthy controls and PAH patients at baseline. In healthy controls, KP-metabolites correlated with each other, with one exception (lack of correlation between Trp and QA (Figure 4A)). In PAH patients, Trp was not correlated with any other metabolite, while other metabolites still correlated with each other at baseline as well as after PAH therapy for six months and one year (Figure 4B-D).

Significant correlations of KP-metabolite levels with baseline characteristics were observed both in healthy controls and PAH patients (Figure 4E&F). Importantly, in PAH patients, higher levels of Kyn, 3-HK, 3-HA, and KA correlated with higher pulmonary vascular resistance, while higher levels of Kyn and KA correlated with reduced cardiac output and cardiac index, higher levels of QA correlated with a reduced cardiac index. Higher levels of Kyn, 3-HK, QA, KA, and AA correlated with worse functional class, higher levels of Kyn correlated with shorter 6-minute walking distance (Figure 4G). In addition, all KP-metabolites correlated with estimated glomerular filtration rate, and Trp, 3-HK, QA, and AA correlated with high-sensitivity C-reactive protein (Figure 4G), further supporting the potential link between inflammation and KP-metabolism.

Survival analyses

PAH patients were stratified into two groups based on the median level of KP-metabolites measured at baseline. High levels of Kyn (>1.328 µM), 3-HK (>22.71 nM) or QA (>75.23 nM) predicted worse early survival (Breslow Test), while high Kyn levels also predicted worse long-term survival (Log-rank Test, Figure 5). However, there was no difference between patients with low and high levels of Trp, 3-HA, KA, or AA (Figure 5).

Since survivors had lower levels of KP-metabolites at the latest measurement timepoint, which may be associated with a better response to PAH therapy, we compared the survival curves in PAH patients based on the latest available measurement. Again, high levels of Kyn, 3-HK, or QA predicted worse early survival, but also worse long-term survival (Figure 5). In addition, patients with high levels of 3-HA (>8.833 nM), KA (>12.92 nM), or AA (>4.944 nM) had worse early survival, and patients with high levels of 3-HA, or KA also had worse long-term survival (Figure 5).

When considering KP-metabolites as continuous variables, for each 1nM increase in Kyn, 3-HK, QA, or KA was associated with an increased hazard ratio of death (Table 3).

These results indicate that elevations in KP-metabolites are potential predictors of survival for PAH patients, with Kyn being the strongest prognostic biomarker.

Table 3

Cox proportional hazard analyses for death by 1nM increase in KP-metabolite levels in PAH patients
	Baseline measurement		Latest measurement
Univariable	Hazard ratio [95% CI]	P value	Hazard ratio [95% CI]	P value
Kyn	1.001 [1.000-1.002]	0.012	1.001 [1.000-1.002]	0.001
3-HK	1.020 [1.009-1.030]	<0.001	1.021 [1.010-1.032]	<0.001
QA	1.006 [1.002-1.010]	0.001	1.007 [1.003-1.011]	0.001
KA	1.045 [0.996-1.096]	0.074	1.054 [1.007-1.104]	0.025
3-HA	1.119 [1.001-1.251]	0.048	1.114 [0.992-1.250]	0.067
AA	1.015 [0.906-1.138]	0.798	1.085 [0.987-1.193]	0.093

Kyn: kynurenine, 3-HK: 3-hydroxy-kynurenine, QA: quinolinic acid, KA: kynurenic acid, 3-HA: 3-hydroxykynurenic acid, AA: anthranilic acid.

The present study demonstrated that 1) KP-metabolism was activated in treatment-naïve PAH patients compared with healthy controls, 2) the KP-metabolite profile in MCT rats was similar to that in PAH patients and was accompanied by an increase in NAD⁺ levels in the rat lungs, 3) exposure of cultured MVECs, PASMCs, and fibroblasts to IL-6/IL-6Rα mimicked the KP-metabolite profile in PAH patients and therefore may contribute to KP activation in PAH patients, 4) PAH therapy normalized KP-metabolite levels in survivors, 5) KP-metabolites correlated with PAH severity at baseline and predicted mortality of PAH patients, particularly when using the latest measurement timepoint.

KP-metabolism is associated with response to PAH therapy and survival

This study is the first to measure the complete KP-metabolite profile in treatment naïve PAH patients and to repeat this measurement after six months and one year of PAH therapy. The results showed that PAH therapy normalizes levels of KP-metabolites in survivors after one year of therapy, which may explain why other PAH cohorts, with patients already undergoing PAH therapy, showed unchanged 3-HK, KA, and AA as compared to controls[14, 16]. This suggests that PAH patients in these cohorts responded to PAH therapy.

Our results further demonstrated that KP-metabolites reflect disease severity in PAH patients, with higher levels of KP-metabolites correlating with higher pulmonary vascular resistance, reduced cardiac index, shorter 6-minute walking distance and/or worse NYHA class. Importantly, survival analysis showed that high levels of KP-metabolites (Kyn, 3-HK, QA, 3-HA, and KA) were strongly associated with worse mortality even after adjusting for age. These results are in accordance with recent studies showing that higher levels of Kyn, 3-HK, and QA correlated with worse functional capacity and mortality in patients with heart failure[24, 25]. Altogether, our data suggest that the KP-metabolite profile can be used as prognostic biomarker for PAH patients. However, detailed analysis of lung tissue obtained from PAH patients should be performed to investigate whether the expression of the enzymes involved in the production of the KP-metabolites is altered in the lungs.

Potential mechanisms underlying the altered KP-metabolism

Immune dysregulation, inflammation and hypoxia are important factors that contribute to the development and progression of PAH[4]. KP-metabolites correlated with high-sensitivity C-reactive protein, a biomarker of inflammation and predictor of outcome in PAH[26]. Since inflammation can decrease eGFR[27], the association between KP-metabolites and eGFR may also represent a common consequence of inflammation. A link between KP-metabolites and inflammation is further supported by our observation that the MCT-PH rats, a model with an obvious inflammatory phenotype, showed a similar KP-metabolite profile as seen in PAH patients. Furthermore, it has been reported that inflammation in PAH patients is alleviated with PAH therapy in survivors[26], which coincides with the normalization of KP-metabolite levels.

To further investigate the inflammatory mediator(s) underlying the KP activation, lung microvascular endothelial cells, smooth muscle cells and fibroblasts were exposed to a variety of inflammatory stimuli, such as IL-6, TNF-α, TGF-β and hypoxia. This in vitro study showed that particularly activation of IL-6/IL-6Rα signaling, which plays a prominent role in pulmonary vascular remodeling in PAH[28], is an important modulator of KP-metabolism. Indeed, IL-6 was elevated in PAH-CTD as well as in a subgroup of iPAH patients in our cohort [29]. Recently, PASMCs were found to be the local source of increased IL-6 in PAH[30], while increased IL-6Rα has been shown in the MVECs[28]. Although the activation of IL-6/IL-6Rα pathway has been shown in both MCT and SuHx[28], the KP-metabolite profile differed markedly between human PAH/MCT and SuHx. This difference may in part be explained by the observed reduction in Kyn levels in the different lung cell types in response to hypoxia. Nevertheless, treatment with tocilizumab, an IL-6R antagonist tested in the TRANSFORM-UK phase-II trial[31], did not result in significant clinical improvement in PAH patients[32] in spite of one case-report showing that tocilizumab improved symptoms in a patient with PAH associated with Castleman’s disease[33]. Since patients recruited in this trial were stable on therapy, it is possible that KP-metabolite levels had normalized, and hence that tocilizumab had no further effects, which might explain the neutral results. It would be interesting to measure the KP-metabolites prior to initiation and post treatment with tocilizumab to identify potential subgroups that might respond to tocilizumab and to further test a causal role of IL-6/IL-6Rα in KP activation.

Can KP activation contribute to progression of PAH?

Although the KP is best known for its link with inflammation[6, 13], its metabolites have a wide variety of other functions. For example, Kyn is a vasodilator activating production cAMP and cGMP / soluble guanylyl cyclase in the systemic[34, 35], coronary[35], as well as pulmonary circulation[15]. Moreover, acute administration of exogenous Kyn reduced right ventricular systolic pressure in rodent models of PH[15]. If vasodilation induced by endogenous Kyn would be important in PAH, higher levels of Kyn should be associated with lower systemic blood pressure and/or lower pulmonary artery pressure. However, although PAH patients had higher levels of Kyn, Kyn correlated with higher PVR with no association with lower systemic or pulmonary artery pressure, and higher levels of Kyn correlated with higher pulmonary artery pressure in previous studies[14, 15]. Therefore, a potential detrimental effect of KP activation that explains its association with worse prognosis in PAH may be mediated through other KP-metabolites.

NAD⁺, the end product of KP-metabolism, plays an essential role in regulation of mitochondrial function and has been implicated in aging/longevity[8]. Several studies suggest that NAD⁺ depletion is an important contributor to the pathogenesis of age-related diseases and cardiovascular diseases[8–10, 36], and raising NAD⁺ levels has been proposed as a promising therapeutic strategy for diseases such as obesity, renal diseases and heart failure[5, 7, 37, 38] by improving mitochondrial function, and prolonging survival of injured cells or apoptotic cells[5, 39, 40]. In contrast to these diseases, PAH is a disease in which mitochondrial remodeling[3] is associated with excessive survival and proliferation of pulmonary vascular cells[4]. Thus, PAH seems to be associated with NAD⁺ abundancy that may contribute to pulmonary vascular remodeling. Indeed, nicotinamide phosphoribosyltransferase (NAMPT), the enzyme responsible for NAD⁺ synthesis via the salvage pathway, was increased in the circulation and lung of advanced PAH patients, and NAMPT inhibition has shown therapeutic effects in rodent models of PH by reversing pulmonary vascular remodeling[11]. In our study, higher levels of KP-metabolites also suggest an activation of de novo NAD⁺ synthesis in PAH patients, particularly since NAD⁺ levels were elevated in the lungs of MCT-PH rats that showed a highly similar KP-metabolite profile. Unfortunately, there was no lung tissue available to measure NAD⁺, and confirm rat data that activation of the KP is indeed associated with higher NAD⁺ levels. Nevertheless, available data suggest that KP activation might contribute to pulmonary vascular remodeling of PAH via de novo NAD⁺ synthesis, future studies should investigate whether inhibition of de novo NAD⁺ synthesis may provide a novel therapeutic target for PAH.

Activation of kynurenine pathway with increased levels of circulating KP-metabolites predict disease severity, response to PAH therapy, and survival in PAH patients. Exposure of lung microvascular endothelial cells, smooth muscle cells and fibroblasts to IL-6/IL-6Rα induced a KP-metabolite profile similar to that observed in PAH patients, suggesting that IL-6/IL-6Rα signaling likely contribute to KP activation in PAH patients. Whether KP activation contributes to pulmonary vascular remodeling via de novo NAD⁺ synthesis, and may provide a novel treatment strategy for PAH remains to be established.

Funding

This work was supported by the China Scholarship Council (201606230252 to ZC & 201708130077 to ST), the China Postdoctoral Science Foundation (2020M680079 and 2021T140598 to ZC), the Netherlands CardioVascular Research Initiative: an initiative with support of the Dutch Heart Foundation (CVON2014-11, RECONNECT, to DM and DJD), and German Center for Cardiovascular Research (DZHK81Z0600207 to DM). Instrumentation support was received from AB Sciex, ltd. for LC-MS/MS analyses performed in this study.

Conflicts of interest/Competing interests The authors declare that they have no conflict of interest

Availability of data and material (data transparency): The dataset supporting the conclusions of this article is included within the article.

Code availability not applicable

Authors' contributions ZC and DM contributed to the conception and design of the work; acquisition, analysis, interpretation of data for the work; drafting and revising the manuscript for the work; final approval of the manuscript to be published. TK, ST, LT, LG, CL, MF, and IK contributed to the acquisition, analysis of data for the work; revising the manuscript for the work; final approval of the manuscript to be published. AB, YR, IR, EB, DD, KB, KL and CG contributed to the interpretation of data for the work, revising of the manuscript for the work; final approval of the manuscript to be published.

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Ethics approval

The (human) study protocol was approved by the Erasmus MC ethical committee and all procedures were performed in accordance with the Declaration of Helsinki. Written informed consent was obtained from all PAH patients and healthy volunteers.

All animal procedures were performed conform to the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes and approved by the respective animal welfare committees of the institute.

Consent to participate (include appropriate statements)

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Simonneau G, Montani D, Celermajer DS, Denton CP, Gatzoulis MA, Krowka M, Williams PG, Souza R: Haemodynamic definitions and updated clinical classification of pulmonary hypertension. Eur Respir J 2019, 53.
Chan SY, Rubin LJ: Metabolic dysfunction in pulmonary hypertension: from basic science to clinical practice. Eur Respir Rev 2017, 26.
Sutendra G, Michelakis ED: The metabolic basis of pulmonary arterial hypertension. Cell Metab 2014, 19:558–573.
Humbert M, Guignabert C, Bonnet S, Dorfmuller P, Klinger JR, Nicolls MR, Olschewski AJ, Pullamsetti SS, Schermuly RT, Stenmark KR, Rabinovitch M: Pathology and pathobiology of pulmonary hypertension: state of the art and research perspectives. Eur Respir J 2019, 53.
Katsyuba E, Mottis A, Zietak M, De Franco F, van der Velpen V, Gariani K, Ryu D, Cialabrini L, Matilainen O, Liscio P, et al: De novo NAD(+) synthesis enhances mitochondrial function and improves health. Nature 2018, 563:354–359.
Minhas PS, Liu L, Moon PK, Joshi AU, Dove C, Mhatre S, Contrepois K, Wang Q, Lee BA, Coronado M, et al: Macrophage de novo NAD(+) synthesis specifies immune function in aging and inflammation. Nat Immunol 2019, 20:50–63.
Yoshino J, Baur JA, Imai SI: NAD(+) Intermediates: The Biology and Therapeutic Potential of NMN and NR. Cell Metab 2018, 27:513–528.
Yaku K, Okabe K, Nakagawa T: NAD metabolism: Implications in aging and longevity. Ageing Res Rev 2018, 47:1–17.
Hershberger KA, Martin AS, Hirschey MD: Role of NAD(+) and mitochondrial sirtuins in cardiac and renal diseases. Nat Rev Nephrol 2017, 13:213–225.
Verdin E: NAD(+) in aging, metabolism, and neurodegeneration. Science 2015, 350:1208–1213.
Chen J, Sysol JR, Singla S, Zhao S, Yamamura A, Valdez-Jasso D, Abbasi T, Shioura KM, Sahni S, Reddy V, et al: Nicotinamide Phosphoribosyltransferase Promotes Pulmonary Vascular Remodeling and Is a Therapeutic Target in Pulmonary Arterial Hypertension. Circulation 2017, 135:1532–1546.
Bender DA: Effects of a dietary excess of leucine on the metabolism of tryptophan in the rat: a mechanism for the pellagragenic action of leucine. Br J Nutr 1983, 50:25–32.
Cervenka I, Agudelo LZ, Ruas JL: Kynurenines: Tryptophan's metabolites in exercise, inflammation, and mental health. Science 2017, 357.
Lewis GD, Ngo D, Hemnes AR, Farrell L, Domos C, Pappagianopoulos PP, Dhakal BP, Souza A, Shi X, Pugh ME, et al: Metabolic Profiling of Right Ventricular-Pulmonary Vascular Function Reveals Circulating Biomarkers of Pulmonary Hypertension. J Am Coll Cardiol 2016, 67:174–189.
Nagy BM, Nagaraj C, Meinitzer A, Sharma N, Papp R, Foris V, Ghanim B, Kwapiszewska G, Kovacs G, Klepetko W, et al: Importance of kynurenine in pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2017, 313:L741-L751.
Jasiewicz M, Moniuszko M, Pawlak D, Knapp M, Rusak M, Kazimierczyk R, Musial WJ, Dabrowska M, Kaminski KA: Activity of the kynurenine pathway and its interplay with immunity in patients with pulmonary arterial hypertension. Heart 2016, 102:230–237.
Galie N, Hoeper MM, Humbert M, Torbicki A, Vachiery JL, Barbera JA, Beghetti M, Corris P, Gaine S, Gibbs JS, et al: Guidelines for the diagnosis and treatment of pulmonary hypertension: the Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS), endorsed by the International Society of Heart and Lung Transplantation (ISHLT). Eur Heart J 2009, 30:2493–2537.
Hoeper MM, Bogaard HJ, Condliffe R, Frantz R, Khanna D, Kurzyna M, Langleben D, Manes A, Satoh T, Torres F, et al: Definitions and diagnosis of pulmonary hypertension. J Am Coll Cardiol 2013, 62:D42-50.
Galie N, Humbert M, Vachiery JL, Gibbs S, Lang I, Torbicki A, Simonneau G, Peacock A, Vonk Noordegraaf A, Beghetti M, et al: 2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT). Eur Heart J 2016, 37:67–119.
Geenen LW, Baggen VJM, Koudstaal T, Boomars KA, Eindhoven JA, Boersma E, Roos-Hesselink JW, van den Bosch AE: The prognostic value of various biomarkers in adults with pulmonary hypertension; a multi-biomarker approach. Am Heart J 2019, 208:91–99.
Menting ME, McGhie JS, Koopman LP, Vletter WB, Helbing WA, van den Bosch AE, Roos-Hesselink JW: Normal myocardial strain values using 2D speckle tracking echocardiography in healthy adults aged 20 to 72 years. Echocardiography 2016, 33:1665–1675.
Tu L, Desroches-Castan A, Mallet C, Guyon L, Cumont A, Phan C, Robert F, Thuillet R, Bordenave J, Sekine A, et al: Selective BMP-9 Inhibition Partially Protects Against Experimental Pulmonary Hypertension. Circ Res 2019, 124:846–855.
Broekhuizen M, Klein T, Hitzerd E, de Rijke YB, Schoenmakers S, Sedlmayr P, Danser AHJ, Merkus D, Reiss IKM: l-Tryptophan-Induced Vasodilation Is Enhanced in Preeclampsia: Studies on Its Uptake and Metabolism in the Human Placenta. Hypertension 2020, 76:184–194.
Lund A, Nordrehaug JE, Slettom G, Hafstad Solvang SE, Ringdal Pedersen EK, Midttun O, Ulvik A, Ueland PM, Nygard O, Melvaer Giil L: Plasma kynurenines and prognosis in patients with heart failure. PLoS One 2020, 15:e0227365.
Konishi M, Ebner N, Springer J, Schefold JC, Doehner W, Dschietzig TB, Anker SD, von Haehling S: Impact of Plasma Kynurenine Level on Functional Capacity and Outcome in Heart Failure- Results From Studies Investigating Co-morbidities Aggravating Heart Failure (SICA-HF). Circ J 2016, 81:52–61.
Quarck R, Nawrot T, Meyns B, Delcroix M: C-reactive protein: a new predictor of adverse outcome in pulmonary arterial hypertension. J Am Coll Cardiol 2009, 53:1211–1218.
Mihai S, Codrici E, Popescu ID, Enciu AM, Albulescu L, Necula LG, Mambet C, Anton G, Tanase C: Inflammation-Related Mechanisms in Chronic Kidney Disease Prediction, Progression, and Outcome. J Immunol Res 2018, 2018:2180373.
Tamura Y, Phan C, Tu L, Le Hiress M, Thuillet R, Jutant EM, Fadel E, Savale L, Huertas A, Humbert M, Guignabert C: Ectopic upregulation of membrane-bound IL6R drives vascular remodeling in pulmonary arterial hypertension. J Clin Invest 2018, 128:1956–1970.
Koudstaal T, van Uden D, van Hulst JAC, Heukels P, Bergen IM, Geenen LW, Baggen VJM, van den Bosch AE, van den Toorn LM, Chandoesing PP, et al: Plasma markers in pulmonary hypertension subgroups correlate with patient survival. Respir Res 2021, 22:137.
Simpson CE, Chen JY, Damico RL, Hassoun PM, Martin LJ, Yang J, Nies M, Griffiths M, Vaidya RD, Brandal S, et al: Cellular sources of IL-6 and associations with clinical phenotypes and outcomes in PAH. Eur Respir J 2020.
Hernandez-Sanchez J, Harlow L, Church C, Gaine S, Knightbridge E, Bunclark K, Gor D, Bedding A, Morrell N, Corris P, Toshner M: Clinical trial protocol for TRANSFORM-UK: A therapeutic open-label study of tocilizumab in the treatment of pulmonary arterial hypertension. Pulm Circ 2018, 8:2045893217735820.
Toshner M, Church C, Morrell N, Corris P: Report: A phase II study of tocilizumab in group 1 pulmonary arterial hypertension. 2018, 17 July.
Arita Y, Sakata Y, Sudo T, Maeda T, Matsuoka K, Tamai K, Higuchi K, Shioyama W, Nakaoka Y, Kanakura Y, Yamauchi-Takihara K: The efficacy of tocilizumab in a patient with pulmonary arterial hypertension associated with Castleman's disease. Heart Vessels 2010, 25:444–447.
Sakakibara K, Feng GG, Li J, Akahori T, Yasuda Y, Nakamura E, Hatakeyama N, Fujiwara Y, Kinoshita H: Kynurenine causes vasodilation and hypotension induced by activation of KCNQ-encoded voltage-dependent K(+) channels. J Pharmacol Sci 2015, 129:31–37.
Wang Y, Liu H, McKenzie G, Witting PK, Stasch JP, Hahn M, Changsirivathanathamrong D, Wu BJ, Ball HJ, Thomas SR, et al: Kynurenine is an endothelium-derived relaxing factor produced during inflammation. Nat Med 2010, 16:279–285.
Zhang D, Hu X, Li J, Liu J, Baks-Te Bulte L, Wiersma M, Malik NU, van Marion DMS, Tolouee M, Hoogstra-Berends F, et al: DNA damage-induced PARP1 activation confers cardiomyocyte dysfunction through NAD(+) depletion in experimental atrial fibrillation. Nat Commun 2019, 10:1307.
Walker MA, Tian R: Raising NAD in Heart Failure: Time to Translate? Circulation 2018, 137:2274-2277.
Canto C, Houtkooper RH, Pirinen E, Youn DY, Oosterveer MH, Cen Y, Fernandez-Marcos PJ, Yamamoto H, Andreux PA, Cettour-Rose P, et al: The NAD(+) precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity. Cell Metab 2012, 15:838–847.
Rajman L, Chwalek K, Sinclair DA: Therapeutic Potential of NAD-Boosting Molecules: The In Vivo Evidence. Cell Metab 2018, 27:529–547.
Poyan Mehr A, Tran MT, Ralto KM, Leaf DE, Washco V, Messmer J, Lerner A, Kher A, Kim SH, Khoury CC, et al: De novo NAD(+) biosynthetic impairment in acute kidney injury in humans. Nat Med 2018, 24:1351–1359.

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Kynurenine Metabolites Predict Survival in Pulmonary Arterial Hypertension: A Role for IL-6/IL-6Rα

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Abstract

Figures

Introduction

Methods

Study population

Animal models of PH

Human and rat EDTA-plasma samples

In vitro study of cultured human primary lung cells

Measurement of KP-metabolites

Statistical analysis

Results

Characteristics of the study population

KP-metabolite profile in PAH

IL-6/IL-6Rα contributed to the activation of KP-metabolism in vitro

Effects of PAH therapy on KP-metabolism

Correlation of KP-metabolites and disease-severity

Survival analyses

Discussion

KP-metabolism is associated with response to PAH therapy and survival

Potential mechanisms underlying the altered KP-metabolism

Can KP activation contribute to progression of PAH?

Conclusion And Clinical Relevance

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1