Metabonomics profiling reveals biochemical pathways associated with

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Metabonomics profiling reveals biochemical pathways associated with pulmonary arterial hypertension in broiler chickens Feng-Jin Shao, Yi-Tian Ying, Xun Tan, Qiao-Yan Zhang, and Wen-Ting Liao J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00316 • Publication Date (Web): 04 Sep 2018 Downloaded from http://pubs.acs.org on September 4, 2018

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Journal of Proteome Research

Metabonomics profiling reveals biochemical pathways associated with pulmonary arterial hypertension in broiler chickens Feng-Jin Shao†, Yi-Tian Ying†, Xun Tan†*, Qiao-Yan Zhang†, Wen-Ting Liao† †

Department of Veterinary Medicine, Zhejiang University, Hangzhou 310058, P.R. China

* Corresponding Author: Dr. Xun Tan, Department of Veterinary Medicine, Zhejiang University,

Hangzhou

310058,

China.

Phone:

+86-571-88982647. E-mail: [email protected].

ORCID ID: 0000-0002-0897-9052

1

ACS Paragon Plus Environment

+86-571-88982393.

Fax:

Journal of Proteome Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ABSTRACT: Pulmonary arterial hypertension (PAH) is the major cause of death in fast growing meat-type chickens (broiler chickens). At present, the underlying mechanisms that give rise to PAH are not fully understood. In order to identify the metabonomics profiles characterizing the process, we conducted a comprehensive gas chromatography-mass spectrometry (GC-MS)-based metabolic profiling of lung tissues from PAH broilers and age-matched controls. PAH was induced by excess salt in drinking water. Medial hypertrophy of pulmonary arteries was present in PAH birds as compared with controls. The metabonomics profiles of lung tissues well distinguished PAH broilers from control subjects. Significant changes in the levels of 41 metabolites were detected in PAH vs. normal birds. Aside from the metabolic alterations indicating a status of oxidative stress and inflammation, evidence of reduced cellular uptake of arginine due to increased lysine biosynthesis and of a shift of arginine metabolism to arginase pathway were observed. In addition, PAH birds showed increased biosynthesis of fatty acids, which may be associated with excessive proliferation of vascular cells during pulmonary vascular remodeling. Furthermore, we observed significant changes in pentose phosphate pathway (PPP) and increased aminomalonic acid in PAH broilers. These results provide additional biochemical insights into the pathogenesis of the PAH. Our data may lead to the development of new strategies to control PAH in broilers.

KEYWORDS: pulmonary arterial hypertension; broiler chicken; lung; metabonomics; oxidative stress; inflammation; nitric oxide; fatty acid; pentose phosphate pathway 2


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INTRODUCTION Fast growing meat-type chickens (broiler chickens) are susceptible to pulmonary arterial hypertension (PAH, also known as ascites syndrome and pulmonary hypertension syndrome), a disease characterized by increased pulmonary arterial pressure leading to right ventricle failure and death. An international survey in commercial broiler flocks showed that PAH affected nearly 5% of broilers 1, making it one of the major causes of death in the broiler industry. A considerable body of evidence suggests that the development of PAH results from increased pulmonary vascular resistance

2-5

. Cumulative evidence attributes the

increased pulmonary vascular resistance to inadequacy of pulmonary vascular capacity to accommodate ever-increasing cardiac output required to meet oxygen demands for rapid growth vasodilators

6, 7

, and to imbalance between vasoconstrictors and

8-15

. Structural remodeling of small pulmonary arteries characterized by

medial hypertrophy and intimal hyperplasia also leads to increased vascular resistance 16-19

. Plexogenic arteriopathy, which might result from dysfunction of endothelial

progenitor cells (EPCs), has been recently implicated in the pathogenesis of PAH

20,

21

. In addition, several lines of evidence suggest that the onset of PAH is associated

with oxidative stress

22-24

and environmental and genetic factors 25. In general, these

studies have provided rich data sets on PAH. However, the exact pathogenic mechanisms underlying the disease still need to be elucidated.

3



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Metabonomics is able to provide a comprehensive view of the changes of specific small-molecule metabolites in biological systems in response to pathophysiological stimuli or genetic modification. As the final downstream products of gene transcription, metabolites reflect the actual functional endpoints of biological events associated with a physiological or pathological state of the cell, tissue, organ or organism. Thus, metabonomics is increasingly being used to investigate disease mechanisms and to detect and identify novel drug targets

26, 27

. Previous studies have

identified altered metabolites in the liver and serum of PAH birds when compared to healthy subjects 28-30. In this study, we aimed at characterizing the specific metabolites in the lung that discriminate PAH birds from healthy controls, to gain new insights into the mechanisms underlying PAH and to identify possible pathways with potential therapeutic value. MATERIALS AND METHODS All experimental procedures were carried out in accordance with the Guidelines of Animal Ethic’s Committee of the Zhejiang University (Approval number ZJU2015-445-12). Animal Management and Sampling

Sixty 1-day-old broiler chickens (Ross-308) were purchased from a local hatchery farm. On day 7, the birds were randomly divided into two groups (n = 30) and one group was subjected to drinking water containing 0.5% (w/v) salt (sodium chloride) to accelerate the occurrence of PAH. During the study standard brooding temperatures 4


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were applied to birds with a 24-h constant light schedule. All chickens had free access to water and were fed a corn/soybean meal-based feed formulated to conform or exceed the minimum National Research Council

31

standards for all ingredients

including 21% CP and 3100 kcal/kg ME. From day 7 on, birds were checked daily for signs of disease and those displaying depression, gasping, cyanosis or/and extended abdomens were humanly killed by cervical dislocation. Hearts were dissected, and right ventricle (RV) and total ventricle (TV) were weighed to calculate RV/TV weight ratio as previously described

20

. A bird having an RV/TV ratio ≥0.28

21

was

considered to suffer from sustained PAH. The whole left lung was removed immediately and cut in the transverse plane at the major rib indentations (costal sulci). One interrib division from the middle of each lung was immediately frozen and stored at −196°C in liquid nitrogen for metabonomics analysis. For histological study, the apical regions of all the sampled lungs were fixed in neutral buffered formalin. Once a PAH bird in the salt-treated group was identified, a bird from the control group was killed, the RV and TV were weighed, and the lung tissue was sampled as described above. Morphometric Analyses The paraffin-embedded lung tissues were cut at 4‒5 µm and stained with Verhoeff’s stain to facilitate the identification of internal and external elastic laminae of muscular pulmonary arteries. The total vessel wall area (TA), i.e., the area within the external elastic lamina, the area within the internal elastic lamina, and mean external and inner 5



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diameter were measured using Image-Pro Plus software (Version 6.0, Media Cybernetics, Rockville, MD). The vessel wall area (WA), WA/TA as well as the relative medial thickness (RMT, medial thickness/external radius) was calculated. Only vessels with complete elastic laminas were evaluated. The differences in morphological parameters were analyzed by two-tailed student’s t-test using the SPSS software (version 22; IBM Corp., Armonk, NY). The significance level was set at α =0.05. Metabolite Extraction Lung tissue sample (50 mg) was homogenized in a mixture of 1 mL chloroform/methanol/water solvent (v/v/v =2:5:2) in an ice bath. Following centrifugation at 14000 g for 15 min at 4 °C, a total of 800 µL supernatant was collected. For an additional extraction, 600 µL of ice-cold methanol was added to the pellet followed by centrifugation at 14000 g, 4 °C for 15 min. A 100 µL of combined supernatants and 10 µL of internal standards (0.05 mg/mL of

13

C6-L-leucine and

13

C6-15N-L-isoleucine) were mixed, dried under a stream of dry nitrogen gas, and

dissolved in 30 µL of 20 mg/mL methoxyamine hydrochloride in pyridine. The resulting mixture was incubated at 37 °C for 90 min. A 30 µL BSTFA (N,O-Bis(trimethylsilyl)trifluoroacetamide) with 1% TMCS (trimethylchlorosilane) (Sigma-Aldrich) was added into the mixture and derivatized at 70 °C for 60 min prior to metabonomics analysis. Quality control (QC) sample pooled from all lung samples

6


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were prepared and analyzed with the same procedure as those of the experiment samples. GC-MS Analysis The derivatized samples were analyzed on an Agilent 7890A gas chromatography system coupled to an Agilent 5975C inert MSD system (Agilent Technologies Inc., CA, USA). A Rxi-5Sil MS fused-silica capillary column (30 m × 0.25 mm × 0.25 µm; Restek corporation, Bellefonte, PA, USA) was used to separate the derivatives. Helium (>99.999%) was used as a carrier gas at a constant flow rate of 1 mL/min through the column. Injection volume was 1 µL, and the solvent delay time was 6 min. The initial oven temperature was held at 70 °C for 2 min, ramped to 160 °C at a rate of 6 °C/min, to 240 °C at a rate of 10 °C/min, to 300 °C at a rate of 20 °C/min, and finally held at 300 °C for 6 min. The temperatures of injector, transfer line, and electron impact ion source were set to 250 °C, 260 °C, and 230 °C, respectively. The impact energy was 70 eV, and data was collected in a full scan mode (m/z 50‒600). Data Processing and Statistical Multivariate Data Analysis Data pre-processing, including the peak picking, alignment and deconvolution, and further processing of raw GC-MS data were performed as previously described

32

.

The resulting data were normalized against total peak abundances and imported to SIMCA software 13.0 package (version 13.0; Umetrics, Umeå, Sweden), where the data were pre-processed by unit variance (UV) scaling and mean centering. Principal component analysis (PCA) and orthogonal partial least squares-discriminant analysis 7



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(OPLS-DA) were applied to pre-processed metabolite concentration data to discriminate spectra of PAH group from control group. In order to avoid model over-fitting, a default 7-round cross-validation in SIMCA software was performed throughout to determine the optimal number of principal components. Potential differential metabolites were identified according to the variable importance in the projection (VIP) values obtained from the OPLS-DA model and the p-value from a two-tailed Student’s t-test of the normalized peak area, and those variables with VIP >1.0 and P 5. Hence, nine metabolic pathways were detected as potential metabolic pathways for PAH and control group (Figure 4). Among these pathways, six biological modules were involved in amino acid metabolism, including D-glutamine and D-glutamate metabolism, alanine, aspartate and glutamate metabolism, glycine, serine and threonine metabolism, lysine biosynthesis, arginine and proline metabolism, and glutathione metabolism. The other three biological modules included arachidonic acid metabolism, pentose phosphate pathway (PPP), and biosynthesis of unsaturated fatty acids. The glycine, serine and threonine metabolism and arginine and proline metabolism were significantly altered (p 5 are labeled. –log (p) is from the original P-value calculated from the enrichment analysis. The x-axis represents the pathway impact, and y-axis represents the pathway enrichment. Larger sizes and darker colors represent higher pathway impact values and higher pathway enrichment.

DISCUSSION To the best of our knowledge, this is the first study to identify lung metabolic abnormalities associated with the pathophysiological mechanism of PAH in broilers. Previous study by Espinha et al.

36

provided evidence that gender has no effect on

cardiorespiratory function or metabolic rate in broilers under resting conditions; however, it appears that their metabolic state changes with age36, 37. To minimize the effect of age on metabolic variables, birds in a narrow age range (between day 10 and 14) were selected for investigation in this work. In addition, PAH birds and controls were individually matched on age. GC-MS analysis revealed a wide range of discriminant metabolites between PAH birds and non-PAH controls, which are related to amino acid metabolism, energy metabolism (PPP, biosynthesis of unsaturated fatty acids) and inflammatory response-related metabolism (arachidonic acid metabolism). These results suggest that PAH pathogenesis may be largely mediated at the level of metabolism.

18


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Insufficient production of nitric oxide (NO), a potent vasodilator in the lung circulation, has been recognized as the major contributor to the pathobiology of PAH in broilers

12, 13, 17, 38

. Nevertheless, the underlying mechanisms still remain unclear.

NO is produced by nitric oxide synthase (NOS) from the amino acid arginine. Thus, any factor that affects the bioavailability of arginine for NOS can lead to reduced synthesis of NO. In the present study, we found that PAH broilers had significantly increased lysine in their lungs, which is in line with a previous study on a rat model of monocrotaline (MCT)-induced PAH cellular uptake of arginine

39

. Lysine is known to competitively inhibit

40, 41

. Given that the availability of arginine is one of the

rate-limiting factors in cellular NO production

42

, and that the transport systems for

lysine and arginine in birds are similar to those reported in mammals

43

, our results

allow us to argue that reduced NO production in the lungs of PAH birds is associated with increased lysine. Indeed, there is evidence that lysine perfusion reduces NO production and increases pulmonary vascular resistance in the lungs isolated from a rat model of lung injury 44. Aside from change in lysine that might lead to reduced NO production, shunting of arginine into a metabolic pathway involving arginase was also evident as arginase products ornithine and urea were significant increased in our PAH broilers. Arginase is known to compete with NOS for arginine, and increased arginase activity may lead to deficiency of arginine available for NOS

45

. Supporting our findings in PAH

broilers, increased breakdown of arginine by arginase was also observed in human 19



PAH

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46

. Inhibition of arginase has been shown to attenuate many of the

pathophysiological symptoms associated with hypoxic pulmonary hypertension

47, 48

.

In context, diversion of arginine to the arginase pathway should also be taken into account for reduce NO production in PAH broilers. Clearly further studies are warranted to test whether PAH birds have increased lung arginase activity. Previous studies on human patients with severe PAH suggest an abnormality in glucose metabolism characterized by reduced glycolysis in the lung

49

. However, in

PAH broilers, we did not determine a significant alteration in glycolysis. Indeed, metabonomics analysis suggested a clear signature of deregulated PPP, as evidenced by a significant decrease in ribose-5-phosphate. Threitol, an end product of xylose metabolism connected to the PPP through the glucuronate pathway, was also down-regulated in our PAH birds. PPP branches out from glycolysis at the first committed step of glucose metabolism, which is required for the synthesis of ribonucleotides and is a major source of nicotinamide adenine dinucleotide phosphate (NADPH). NADPH is essential for anabolic reactions and redox homeostasis maintenance 50. Thus, decrease in PPP flux may explain the increased oxidative stress in PAH 22, 51. Glutathione (GSH), which is synthesized from its precursor amino acids glycine, cysteine, and glutamic acid, plays a key role in cellular response against oxidative stress. In this work, we did not detect an alteration of GSH in PAH lungs. However, the levels of 2-aminobutyric acid and 2-hydroxybutyric acid, which are generated as 20


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byproducts when cystathionine is cleaved to cysteine in times of high GSH demand under oxidative stress

52

, were markedly elevated. It is suggested that increased

2-aminobutyric acid compensates GSH level for consumption by reactive oxidative species (ROS) and promotes GSH synthesis

52

. In this context, the increases in

2-aminobutyric acid and 2-hydroxybutyric acid likely reflect increased oxidative stress in PAH lungs. We also determined decreased levels of glutamic acid as well as O-phosphoserine, the precursor of cysteine, in PAH birds, which may be a consequence of enhanced demand of GSH biosynthesis. In addition, PAH group demonstrated a substantial increase in aminomalonic acid. The origin of aminomalonic acid is associated with free radical damage to proteins, mainly glycine and cysteine

53

. In mammals, increased aminomalonic acid was

observed in cardiovascular diseases with an oxidative stress component, including human PAH

54

. Taken together, our results are consistent with the concept that

oxidative stress is involved in the development of PAH. In the present study, PAH broilers exhibited significant alterations in lipid metabolic activity in their lungs as evident by increased amounts of fatty acids. This metabolic shift is likely associated with excessive proliferation of vascular cells during pulmonary artery remodeling, since hyperproliferating cells have high rates of de novo fatty acid synthesis critical for membrane formation and energy production 55. It has been shown that the expression and activity of fatty acid synthase, an enzyme important for fatty acid synthesis, are increased in proliferating pulmonary smooth 21



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muscle cells and endothelial cells, and in the lungs of pulmonary hypertensive rats; accordingly, inhibition of fatty acid synthesis reverses pulmonary vascular remodeling and endothelial dysfunction associated with PAH

56, 57

. Although there is a lack of

direct evidence that fatty acids are involved in pulmonary vascular remodeling in PAH broilers, enhanced PAH mortality in birds fed diets containing higher levels of either saturated or unsaturated fats has been previously observed

58

. Further studies

are required to determine whether the fatty acid synthase plays a role in broiler PAH. Growing evidence suggests that inflammation plays a significant role in broiler PAH

59

. In the present study, increased level of arachidonic acid was found in the

lungs of PAH broilers when compared with healthy subjects. Arachidonic acid pathway is a central regulator of inflammatory response. Arachidonic acid is synthesized from α-linolenic acid derived from linoleic acid, then is converted by lipooxygenase into leukotrienes (LTs) or by cyclooxygenases into prostaglandin H2 (PGH2), which is further processed by a series of specific isomerase and synthase enzymes to produce various eicosanoids including prostaglandin D2, prostaglandin J2, and prostaglandin E2. The formation of leukotrienes and prostaglandins from AA constitutes an inflammatory microenvironment that attracts and stimulates leukocytes 60

. To this end, arachidonic acid might be considered as a biomarker of inflammation

and a therapeutic target of PAH. CONCLUSIONS

22


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In summary, this study investigated the metabolic signatures of the PAH broiler lung using an untargeted metabonomics platform. Results from this global metabolic profiling study revealed number of metabolic alterations that might account for impaired NO production, pulmonary vascular remodeling, oxidative stress and inflammation in PAH birds. We propose, for the first time, the occurrence of PAH in broilers is associated with deregulated PPP. The results of this study may lead to the development of new strategies to treat PAH in broilers.

AUTHOR INFORMATION

Corresponding Author: Dr. Xun Tan, Department of Veterinary Medicine, Zhejiang University, 866 Yuhantang Road, Hangzhou 310058, China; Phone: +86-571-88982393; Fax: +86-571-88982647; E-mail: [email protected]. ORCID ID: 0000-0002-0897-9052 Author Contributions The manuscript was written through contributions of all authors. Feng-Jin Shao reared the chickens, collected samples and did lab analyses. Yi-Tian Ying contributed to data statistical analysis and manuscript writing. Xun Tan conceived the idea, supervised the experiment, lab analyses and data statistical analysis and wrote the manuscript. Qiao-Yan Zhang contributed to chicken management and sample

23



collection. Wen-Ting Liao contributed to sample collection. All authors have given approval to the final version of the manuscript. CONFLICT OF INTEREST DISCLOSURE The authors declare no competing financial interest. ACKNOWLEDGMENT This work was supported by Zhejiang Provincial Natural Science Foundation of China (project no. LR12C18001). We would also like to thank Shanghai ProfLeader Biotech Co, Ltd for assistance with the GC-MS analysis. SUPPORTING INFORMATION Table S1. The detailed results of potential metabolic pathways for PAH group vs. control group. Figures S1‒9: KEGG metabolic pathways relevant to PAH. Figure S1. Glutathione metabolism pathway. Figure S2. Glycine, serine and threonine metabolism pathway. Figure S3. Biosynthesis of unsaturated fatty acids. Figure S4. Arginine and proline metabolism. Figure S5. D-Glutamine and D-glutamate metabolism. Figure S6. Alanine, aspartate and glutamate metabolism. Figure S7. Arachidonic acid metabolism pathway. Figure S8. Lysine biosynthesis pathway. Figure S9. Pentose phosphate pathway ABBREVIATIONS

24


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PAH: pulmonary arterial hypertension; RV/TV: right/total ventricle; TA: vessel wall area; WA: wall area; RMT: relative medial thickness; GC-MS: gas chromatography–mass spectrometry; TIC: total ion chromatogram; PCA: principal component analysis; OPLS-DA: orthogonal partial least squares-discriminant analysis; PPP: pentose phosphate pathway; NO: nitric oxide; NOS: nitric oxide synthase; GSH: glutathione REFERENCES 1.

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