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De novo synthesize of bile acids in pulmonary arterial hypertension lung

Article  in  Metabolomics · December 2014

DOI: 10.1007/s11306-014-0653-y · Source: PubMed

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Metabolomics (2014) 10:1169–1175
DOI 10.1007/s11306-014-0653-y

SHORT COMMUNICATION

De novo synthesize of bile acids in pulmonary arterial

hypertension lung
Yidan D. Zhao • Hana Z. H. Yun • Jenny Peng •
Li Yin • Lei Chu • Licun Wu • Ryan Michalek •
Mingyao Liu • Shaf Keshavjee • Thomas Waddell •

John Granton • Marc de Perrot

Received: 18 October 2013 / Accepted: 26 March 2014 / Published online: 11 April 2014
Ó The Author(s) 2014. This article is published with open access at Springerlink.com

Abstract Although multiple, complex molecular studies the development of PAH. Here, by profiling the meta-
have been done for understanding the development and bolomic heterogeneity of the PAH lung, we reveal a newly
progression of pulmonary hypertension (PAH), little is discovered pathogenesis mechanism of PAH.
known about the metabolic heterogeneity of PAH. Using a
combination of high-throughput liquid-and-gas-chromato- Keywords Bile acid pathway
Pulmonary arterial
graphy-based mass spectrometry, we found bile acid hypertension
Lung
metabolites, which are normally product derivatives of the
liver and gallbladder, were highly increased in the PAH
lung. Microarray showed that the gene encoding cyto- 1 Introduction
chrome P450 7B1 (CYP7B1), an isozyme for bile acid
synthesis, was highly expressed in the PAH lung compared Pulmonary arterial hypertension (PAH) is a severe vascular
with the control. CYP7B1 protein was found to be pri- disease characterized by persistent precapillary pulmonary
marily localized on pulmonary vascular endothelial cells hypertension (PH) (Stacher et al. 2012; International PPHC
suggesting de novo bile acid synthesis may be involved in et al. 2000; Zhao et al. 2002; Fujiwara et al. 2008; Nasim
et al. 2011; Olschewski 2010; Bogaard et al. 2012; MMea
Electronic supplementary material The online version of this and 2013), which can be either be idiopathic (sporadic-
article (doi:10.1007/s11306-014-0653-y) contains supplementary 90 %, familial-10 %). PAH can also be a complication
material, which is available to authorized users. associated with other conditions such as connective tissue
disease, congenital heart disease, anorexigen use (dexfen-
Y. D. Zhao
H. Z. H. Yun
J. Peng
L. Yin
L. Chu
L. Wu

M. Liu
S. Keshavjee
T. Waddell
M. de Perrot fluramine), portal hypertension, and human immunodefi-
Latner Thoracic Surgery Research Laboratories, Division of ciency virus (Stacher et al. 2012; International PPHC, Lane
Thoracic Surgery, University of Toronto, Toronto, ON, Canada KB, Machado RD, Pauciulo MW, Thomson JR, et al. 2000;
MMea et al. 2013). Evidence in the literature suggests that
Y. D. Zhao (&)
M. de Perrot (&)
MaRS Centre, Toronto Medical Discovery Tower, 2nd Floor Rm metabolic pathway abnormalities characterize and may
2-817, 101 College Street, Toronto, ON M5G 1L7, Canada play a significant role in the development and progression
e-mail: yidanzhao@gmail.com of PAH (Fessel et al. 2012). For example, pulmonary
M. de Perrot arterial endothelial cells (PAECs) in PAH share similar
e-mail: marc.deperrot@uhn.ca hyperproliferative characteristics as malignant tumor
transformation that is accompanied by significant meta-
R. Michalek
Metabolon, Incorporated, 617 Davis Drive, Durham, NC 27713, bolic shifts to support anabolic growth and energy metab-
USA olism (Xu et al. 2005; Chen et al. 2007). Moreover, it has
been shown that mitochondrial oxidative phosphorylation
J. Granton
with glucose uptake and utilization occurs in PAEC
Clinical Studies Resource Centre, Toronto General Hospital,
University Health Network, University of Toronto, Toronto, ON, development. Significant elevation of hemoglobin has been
Canada found in the PAH sample group without a history of

123
1170 Y. D. Zhao et al.

RT: 5.04
RT: 4.41 - 5.74 SM: 5G
AA: 100367
BP: 514.4 NL: 6.57E4
100 m/z= 513.8-514.8 F: ITMS
- c ESI Full ms
80 PPH sample [80.00-1000.00] MS ICIS
Relative Abundance

Peak for Taurocholate

LTQ2NEG20120924_UNT

60 O0412_LUNG2_17

40

RT: 5.11
20 RT: 4.62 AA: 1168 RT: 5.27 RT: 5.48 RT: 5.61
RT: 4.89 BP: 514.7 RT: 5.21 AA: 21807 AA: 18581 AA: 9792
AA: 6010 BP: 514.5
AA: 3718 AA: 6265 BP: 514.5 BP: 514.1
BP: 514.6
BP: 514.4 BP: 514.8
0 RT: 5.49
AA: 23892
BP: 514.5 NL: 9.00E3
100
m/z= 513.8-514.8 F: ITMS
NL sample
- c ESI Full ms
80 [80.00-1000.00] MS ICIS
Relative Abundance

RT: 5.60 LTQ2NEG20120924_UNT

AA: 10278
BP: 514.1
60
RT: 4.61 O0412_LUNG1_29
AA: 6823
BP: 514.7

40 No peak RT: 5.24

AA: 9787
BP: 514.7

RT: 5.35 RT: 5.71

RT: 5.09
20 AA: 2357
AA: 1702 AA: 3026
BP: 513.9 BP: 514.3
BP: 514.6

0
4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 5.6 5.7
Time (min)

496.3
100
353.3
PAH sample NL: 7.92E2
80 LTQ2NEG20120924_UNTO04
Relative Abundance

12_LUNG2_17#1262 RT:
5.04 AV: 1 F: ITMS - c ESI d
60 Full ms2 514.38@cid30.00
[130.00-1040.00]

40
351.3 354.4 497.4
369.3
412.2
453.3
20 432.4
495.7

333.4 410.5 476.6

323.2 372.5 451.5 469.3
243.2
167.1 187.1 208.1 216.2 259.3 275.2 302.4 357.7 415.5 500.2
0 496.3
100
353.2
NL: 1.43E4
80 LTQ220071031N_PLEX106_
Relative Abundance

HIGH_41#1342 RT: 5.10 AV:

Authentic standard
1 F: ITMS - c ESI d Full ms2
60 514.25@cid40.00
[130.00-1040.00]

40
371.3
412.3
497.3

329.2 369.3 495.3

20 351.2 430.4
315.3 432.4
414.4
314.2 335.2 372.3 394.3 469.2 493.3
342.7 428.3 450.4 482.3
165.1 178.2 206.1 215.3 241.3 259.2 281.2 300.5 368.3 382.4 498.3
0
160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
m/z

123
 De novo synthesize of bile acids 1171

b Fig. 1 MS/MS fragmentation spectrum of taurocholate in control and 2009). The detail procedure of metabolic analysis has been
PAH lung. Top panel shows a representative negative ion, selected documented in the Supplement data.
ion chromatogram (SIC) for taurocholate (m/z 514.3) in normal (NL)
and pulmonary hypertension (PAH) lung tissue. Taurocholate com-
pound identification relied on confirmed experimental MS/MS 2.1 Transcriptomic analysis
fragmentation spectrum matched to the authenticated taurocholate
standard, run separately (bottom panel). Limited peak detection was mRNA samples from the normal (n = 8) and native PAH
observed in NL samples
lungs (n = 8) were isolated as described (Zhao et al. 2014).
Bile acid related profiles were compared between a control
diabetes or any other obvious metabolic diseases, indicat- group and samples with idiopathic pulmonary arterial
ing the impairment of whole-body glucose homeostasis in hypertension. Briefly, the total RNA analysis in lung tis-
PAH (Pugh et al. 2011; Hansmann et al. 2007; Archer et al. sues was performed using Trizol extraction according to
2010). Additionally, vascular changes under chronic hyp- the manufacturer’s instructions. Biotinylated cRNA was
oxic condition has been directly linked to an imbalance prepared according to the standard Affymetrix protocol
between glycolysis, glucose oxidation, and fatty acid oxi- (Expression Analysis). Following fragmentation, cRNA
dation (Sutendra et al. 2010), while in vitro pulmonary were hybridized on GeneChip Genome Array. GeneChips
arterial endothelial cell culture with disruption of the Bone were scanned using the HuGene-1_0-st-v1 GeneArray
Morphogenetic Protein Receptor II (BMPRII) gene showed Scanner G2500A. The data were analyzed with Partek
significant metabolomic changes (Fessel et al. 2012). Our Genomics Suite 6.6 using the Affymetrix default analysis
recent work showed that disrupted glycolysis, increased settings and global scaling as the normalization method.
TCA cycle, and fatty acid metabolites with altered oxida- The value definition was set up using Partek Genomics
tion pathways exited in the human PAH lung, indicating Suite 6.6. Significantly changed genes were determined by
that PAH has specific metabolic pathways contributing to t test with a false discovery rate of two fold. The data base
abnormal ATP synthesis for the vascular remodeling pro- has been submitted to NCBI/GEO and has been approved
cess in pulmonary hypertension (Zhao et al. 2014). Col- and assigned a GEO accession number GSE53408.
lectively, in vitro, human and animal models suggest that
multiple metabolic pathways are reprogrammed during 2.2 Immunoblotting
PAH vascular remolding and that metabolic heterogeneity
may play an important role in both ATP energy supply and Protein concentrations were determined using the BCA
the molecular pathogenesis of pulmonary hypertension. protein assay (Pierce, IL, USA). Equal amounts of the pro-
Here, we provide direct evidence of a novel increase in bile tein lysates were separated by SDS-PAGE and transferred
acid metabolites in PAH lung tissue associated with the onto nitrocellulose membranes. The membranes were incu-
elevated expression of bile acid synthesis related tran- bated for overnight at 4 °C with the following antibodies
scripts, indicating de novo synthesis of bile acids may from AbcamR: anti-CYP7B1(1:1,000). After wash with
characterize and contribute to the pathogenesis of PAH. TBS-Tween, the blots were incubated for 60 min at room
temperature with horseradish peroxidase-conjugated anti-
bodies, respectively: anti-rabbit antibody (1:15,000; Sigma-
2 Materials and methods Aldrich, St. Louis, MO). Signals from immunoreactive
bands were visualized by fluorography using an ECL reagent
Global biochemical profiles were determined in human (Pierce). The intensity of individual bands in immunoblots
lung tissue and compared across 8 normal (47 ± 15 years were quantified using the NIH Image program.
of age, 4 females) and 8 pulmonary arterial hypertension
patients (40 ± 12 years of age, 5 females). Eligibility cri- 2.3 Immunohistochemistry
teria included end stage PAH patients who went through
lung transplantation. Lung samples were obtained from the The sections of both PAH and normal lung tissue were
recipient lung at the time of lung transplantation. Control fixed for 4 h at room temperature with PBS made of 4 %
lung samples were obtained from normal tissue of cancer formaldehyde, permeabilized for 30 min in Triton X-100
patients undergoing surgery (lobectomy). Biospecimens (0.5 % in PBS), and incubated with 5 % nonfat skim milk
and associated clinical data related to the study were col- in PBS for 90 min. Sections were incubated for 180 min at
lected with written consent from the University Health room temperature with antibodies for anti-CYP7B1
Network and approved by the Internal Review Board. (1:1,000). The sections were then incubated with biotin-
Unbiased metabolomic profiling using liquid/gas chroma- ylated secondary antibody and visualized with DAB.
tography coupled to mass spectrometry (LC/GC–MS) was Stained cells and sections were visualized with the Zeiss
performed as described (Reitman et al. 2011; Evans et al. LSM 510 confocal microscope.

123
1172 Y. D. Zhao et al.

RT: 5.06
RT: 4.48 - 5.67 SM: 5G
AA: 100063 RT: 5.42
BP: 464.4 AA: 230796 NL: 7.06E4
100 BP: 464.3 m/z= 463.9-464.9 F: ITMS
- c ESI Full ms
PAH sample [80.00-1000.00] MS ICIS
Relative Abundance

80 LTQ2NEG20120924_UNT
O0412_LUNG1_09
60
Peak for Glycocholate
RT: 5.56
40 AA: 62313
BP: 464.4

20 RT: 4.63
RT: 5.20
AA: 10135 RT: 4.90
RT: 4.58 BP: 464.4 AA: 3952 AA: 17465
AA: 1223 BP: 464.4 BP: 464.2
BP: 464.2
0 RT: 5.24
AA: 22719
BP: 464.5 NL: 3.05E3
100 RT: 5.06 m/z= 463.9-464.9 F: ITMS
RT: 5.54
AA: 5267 AA: 6129 RT: 5.58 - c ESI Full ms
BP: 464.7 BP: 464.3 AA: 2707 [80.00-1000.00] MS ICIS
BP: 464.3
80 NL sample
Relative Abundance

RT: 5.33 LTQ2NEG20120924_UNT

AA: 2890
BP: 464.1 O0412_LUNG1_29
RT: 5.66
AA: 1694
60 RT: 5.44 BP: 464.4
AA: 2449
BP: 464.6

40

20

0
4.5 4.6 4.7 4.8 4.9 5.0 5.1 5.2 5.3 5.4 5.5 5.6
Time (min)
402.3 NL: 4.67E3
100 LTQ2NEG20120924_UNTO0
412_LUNG1_09#1268 RT:
PAH sample 5.06 AV: 1 F: ITMS - c ESI d
Relative Abundance

80 Full ms2 464.42@cid30.00

[115.00-940.00]
60

40
420.4

403.5 446.3
20
400.3
431.0 447.3
419.4
384.4
147.9 166.1 190.2 198.2 210.1 221.0 237.2 257.2 281.0 294.8 310.4 321.6 341.3 353.3 371.4 389.3 418.3 461.4
0
100 402.4
NL: 6.59E3
LTQ2NEG20080627_PLEX1
80
Authentic standard 09_1_37#1550 RT: 5.45 AV:
1 F: ITMS - c ESI d Full ms2
60 464.39@cid26.00
[115.00-940.00]

40
403.4 446.4

20 420.4 447.4
400.5
431.2 448.6
384.4 419.5 453.9
177.2 193.1 212.2 221.0 244.2 274.4 286.3 295.3 314.9 321.5 353.4 366.2 465.4
0
140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460
m/z

123
 De novo synthesize of bile acids 1173

b Fig. 2 MS/MS fragmentation spectrum of glycocholate in control

and PAH lung. Representative negative ion is selected ion chromato-
a
gram (SIC) for glycocholate (m/z 464.4) in normal (NL) and

Relitive CYP7B1
pulmonary hypertension (PAH) lung tissue (top panel). Glycocholate

expression
compound identification relied on confirmed experimental MS/MS 9.0
fragmentation spectrum matched to the authenticated glycocholate
standard, run separately (bottom panel)
*
8.5

8.0
taurocholate glycocholate
a. 10
8 NL PAH

4 5 b
relative metabolite abundance

0 0
glycolithocholate sulfate mannitol

3 1400
2
700
1
0 0
glycochenodeoxycholate taurochenodeoxycholate
c
10 20 CYP7B1

5 10 GAPDH

0 0
NL PAH NL PAH NL PAH

Fig. 3 PAH lung has a unique bile acids metabolic pathway.

150
CYP7B1 (%)

Intermediates in the bile acids pathway revealed significantly elevated

levels of multiple glycine and taurine conjugated bile acids in the *
PAH lung. Data for normal lung (NL, n = 8) are represented in green 100
boxes, while data for pulmonary hypertension lung (n = 8) are shown
in pink boxes. Quantities are in relative arbitrary units specific to the 50
internal standards for each quantified metabolite and normalized to
protein concentration (PAH with red frame indicates *p \ 0.05 0
compared to NL NL PAH

Fig. 4 a Microarray data showed that the gene encoding cytochrome

3 Results and discussion P450, family 7, subfamily B, polypeptide 1 (Oxysterol 7a-hydrox-
ylase) was significantly highly expressed in PAH lung.
We explored and characterized the metabolomic signature (p = 0.000187299). b Western blot analysis of CYP7B1 expression
in normal and PAH lungs. Lung lysate was loaded and immunoblotted
of pulmonary hypertension (PAH) to enhance our under-
with antibody against CYP7B1 and GAPDH (loading control).
standing of disease progression. Using untargeted meta- Consistent with a significant increase of CYP7B1 gene expression
bolic profiling, we found that PAH lung (n = 8) possessed in PAH, the enzyme protein for CYP7B1 (37KD) was significantly
significantly higher levels of multiple bile acid metabolites, increased in PAH lungs compared with NL lungs. Densitometric
analysis of CYP7B1 was normalized to the intensity of the respective
including the primary bile acids taurocholate (Fig. 1),
GAPDH band. Data are expressed as mean ± SD (n = 4). *p \ 0.05
glycocholate (Fig. 2), taurochenodeoxycholate, and gly- versus NL. c CYP7B1 positive immunostaining in newly formed
cochenodeoxycholate (Fig. 3). Bile acids are normally small blood vessels (arrows) in the plexiform lesions of occluded
synthesized in the liver and gallbladder from cholesterol by pulmonary small vessel in PAH lung. Representative micrographs of
immunostaining of PAH lung sections are shown with anti–CYP7B1
7-alpha-hydroxylase, also called cytochrome P450
in the pulmonary vascular endothelial cells. (ratio 1:200)
(CYP7A1), as a rate-limiting enzyme in the synthesis of
bile acid via the classic pathway (Nishimoto et al. 1993;
Cohen et al. 1992; Crestani et al. 1993; Wang and Chiang surprisingly revealed that the gene encoding cytochrome
1994). Although the presence of bile acids in lung tissue P450 B1 (CYP7B1), but not CYP7A1, had a significantly
may partially reflect reflux in these patient (D’Ovidio et al. higher expression in PAH lung (Fig. 4a). This finding was
2005; Blondeau et al. 2009), microarray analysis also confirmed by Real time RTPCR. Further molecular

123
1174 Y. D. Zhao et al.

cholesterol Blondeau, K., Mertens, V., Vanaudenaerde, B. A., Verleden, G. M.,

Van Raemdonck, D. E., et al. (2009). Nocturnal weakly acidic
reflux promotes aspiration of bile acids in lung transplant
CYP7A1 CYP7B1 recipients. The Journal of Heart and Lung Transplantation, 28,
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Bogaard, H. J., Al Husseini, A., Farkas, L., Farkas, D., Gomez-
7-a-hydroxycholesterol-4-en-3-one Arroyo, J., et al. (2012). Severe pulmonary hypertension: The
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chenodeoxycholate
Warburg effect and its cancer therapeutic implications. Journal
of Bioenergetics and Biomembranes, 39, 267–274.
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CYP7B1enzyme was higher in PAH lung (Fig. 4b). These T., et al. (2012). Metabolomic analysis of bone morphogenetic
protein receptor type 2 mutations in human pulmonary endo-
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solely be due to reflux from the esophagus (D’Ovidio et al. ary Circulation, 2, 201–213.
2005; Blondeau et al. 2009) but come from the lung itself. Fujiwara, M., Yagi, H., Matsuoka, R., Akimoto, K., Furutani, M.,
Thus, PAH lung tissue may have the capacity for de novo et al. (2008). Implications of mutations of activin receptor-like
kinase 1 gene (ALK1) in addition to bone morphogenetic protein
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metabolites could potentially serve as biomarkers for dis- hypertension. Circulation Journal, 72, 127–133.
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Open Access This article is distributed under the terms of the gene encoding human liver cholesterol 7 alpha-hydroxylase.
Creative Commons Attribution License which permits any use, dis- Biochimica et Biophysica Acta, 1172, 147–150.
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author(s) and the source are credited. approach. Advances in Experimental Medicine and Biology, 661,
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