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Published in final edited form as:

Cell Metab. 2024 June 04; 36(6): 1335–1350.e8. doi:10.1016/j.cmet.2024.04.010.

Dietary intake and glutamine-serine metabolism control

pathologic vascular stiffness
Nesrine S. Rachedi1,*, Ying Tang2,3,*, Yi-Yin Tai2,3, Jingsi Zhao2,3, Caroline Chauvet1,
Julien Grynblat4,5, Kouamé F. Akoumia4, Leonard Estephan2,3, Stéphanie Torrino1, Chaima
Sbai1, Amel Ait-Mouffok1, Joseph D. Latoche6, Yassmin Al Aaraj2,3, Frederic Brau1, Sophie
Abélanet1, Stephan Clavel1, Yingze Zhang2,7, Christelle Guillermier8, Naveen VG Kumar9,
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Sina Tavakoli3,10, Olaf Mercier4,11, Michael G. Risbano2,7, Zhong-Ke Yao12, Guangli Yang12,
Ouathek Ouerfelli12, Jason S. Lewis12, David Montani5,11, Marc Humbert4,11, Matthew L.
Steinhauser8,9, Carolyn Anderson13, William M. Oldham14, Frédéric Perros4,15, Thomas
Bertero1,§,#,$, Stephen Y. Chan2,3,§,#
(1)Université Côte d’Azur, CNRS, INSERM, IPMC, IHU-RespirERA Valbonne, France;
(2)Centerfor Pulmonary Vascular Biology and Medicine, Pittsburgh Heart, Lung, and Blood
Vascular Medicine Institute,
(3)Division
of Cardiology, Department of Medicine, University of Pittsburgh School of Medicine and
UPMC, Pittsburgh, PA, USA;
(4)UniversitéParis–Saclay, AP-HP, INSERM UMR_S 999, Service de Pneumologie et Soins
Intensifs Respiratoires, Hôpital de Bicêtre, Le Kremlin Bicêtre, France
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(5)Pôle
Thoracique, Vasculaire et Transplantations, Hôpital Marie Lannelongue, Le Plessis-
Robinson, France;

#
Corresponding Authors: Thomas Bertero, Ph.D., Université Côte d’Azur, Institute of Molecular and Cellular Pharmacology,
CNRS UMR 7275 – INSERM U1323, 660, route des Lucioles, Sophia Antipolis, 06560 Valbonne, France, thomas.bertero@univ-
cotedazur.fr, Stephen Y Chan, M.D., Ph.D., Center for Pulmonary Vascular Biology and Medicine, Pittsburgh Heart, Lung, and
Blood Vascular Medicine Institute, Division of Cardiology, Department of Medicine, University of Pittsburgh School of Medicine and
UPMC, 200 Lothrop Street BST E1240, Pittsburgh, PA USA 15261, chansy@pitt.edu.
*denotes equal contribution by these authors.
§denotes equal contribution by these authors.
$Lead contact
Author contributions: S.Y.C. and T.B. conceived and designed the experiments. S.Y.C., W.M.O., F.P. and T.B., provided
experimental infrastructure. N.R., Y.T., C.C., S.T. C.S., A.A.M., Y-Y.T., J.Z., W.M.O., and T.B. performed the in vitro experiments.
F.B., S.C. and S.A. performed confocal imaging experiments. Y.T., Y-Y.T., J.Z.., J.G., K.F.A., F.P., and S.Y.C. performed the in vivo
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rat and mouse experiments. L.E., Y.T., Y-Y.T., S.Y.C., and M.L.S. performed the MIMS study. G.Y., Z.-K.Y, and O.O. synthesized the
18F-Gln precursor and FGln standard. Y.T., M.T., J.D.L., S.T., S.Y.C., and C.A. performed the PET imaging studies. Y.A.A., M.G.R.,
and Y.Z. provided human plasma samples and clinical phenotyping data. O.M., D.M., F.P., and M.H., provided human PAAF and
clinical phenotyping data. W.M.O. performed and analyzed the metabolomic experiments. S.Y.C. and T.B. wrote the manuscript. All
authors participated in interpreting the results and revising the manuscript.
Declaration of Interests: S.Y.C. has served as a consultant for Merck, Janssen, and United Therapeutics. S.Y.C. is a director, officer,
and shareholder in Synhale Therapeutics. S.Y.C. has held research grants from WoodNext, Bayer, and United Therapeutics. S.Y.C. and
T.B. have filed patent applications regarding the targeting of metabolism in pulmonary hypertension. G.Y. Z.-K.Y and O.O. are listed
as inventors in patents, not related to this work, that are filed by MSKCC. O.O. receives royalties from MSKCC, Johnson & Johnson,
Jazz, and Y-mAbs and owns shares of Angiogenex for which he is an unpaid member of the SAB, all of which are not related to this
work. The other authors declare no competing interests.
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 Rachedi et al. Page 2

(6)Hillman Cancer Center, University of Pittsburgh School of Medicine and UPMC, Pittsburgh, PA,
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USA;
(7)Divisionof Pulmonary and Critical Care Medicine, Department of Medicine, University of
Pittsburgh School of Medicine and UPMC, Pittsburgh, PA, USA;
(8)Centerfor NanoImaging, Division of Genetics, Brigham and Women’s Hospital and Harvard
Medical School, Boston, MA
(9)AgingInstitute, Division of Cardiology, Department of Medicine, University of Pittsburgh School
of Medicine and UPMC, Pittsburgh, PA, USA;
(10)Department of Radiology, University of Pittsburgh School of Medicine and UPMC, Pittsburgh,
PA, USA;
(11)Assistance Publique-Hôpitaux de Paris (AP-HP), Service de Pneumologie et Soins Intensifs
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Respiratoires, Centre de Référence de l’Hypertension Pulmonaire, Hôpital Bicêtre, 94270 Le

Kremlin-Bicêtre, France;
(12)Molecular
Pharmacology and Chemistry Program and Organic Synthesis Core Facility,
Memorial Sloan Kettering Cancer Center, New York, USA;
(13)Department of Chemistry, University of Missouri, Columbia, MO USA;
(14)Division
of Pulmonary and Critical Care Medicine, Department of Medicine, Brigham and
Women’s Hospital, Harvard Medical School, Boston, MA USA
(15)Laboratoire
CarMeN, UMR INSERM U1060/INRA U1397, Université Claude Bernard Lyon1,
F-69310 Pierre-Bénite

Summary
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Perivascular collagen deposition by activated fibroblasts promotes vascular stiffening and drives
cardiovascular diseases such as pulmonary hypertension (PH). Whether and how vascular
fibroblasts rewire their metabolism to sustain collagen biosynthesis remain unknown. Here, we
found that inflammation, hypoxia, and mechanical stress converge on activating the transcriptional
coactivators YAP and TAZ (WWTR1) in pulmonary arterial adventitial fibroblasts (PAAF).
Consequently, YAP and TAZ drive glutamine and serine catabolism to sustain proline and glycine
anabolism and promote collagen biosynthesis. Pharmacologic or dietary intervention on proline
and glycine anabolic demand decreases vascular stiffening and improves cardiovascular function
in PH rodent models. By identifying the limiting metabolic pathways for vascular collagen
biosynthesis, our findings provide guidance for incorporating metabolic and dietary interventions
for treating cardiopulmonary vascular disease.
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Graphical Abstract

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eTOC blurb
Rachedi et al. identify the precise metabolic requirements for pathogenic vascular fibroblast
activation, using pulmonary arterial hypertension as an example. Inflammation, hypoxia, and
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mechanical stress converge on YAP and TAZ activation. YAP and TAZ control glutamine and
serine catabolism to generate proline and glycine for collagen biosynthesis. Pharmacologic or
dietary intervention on those metabolic pathways decreases collagen production and vascular
stiffening and improves pulmonary vascular function.

Keywords
Cardiovascular Disease; Metabolism; Nutrition; Vascular Fibroblast; Fibrosis; Collagen
Metabolism

INTRODUCTION
Cardiac and vascular diseases (CVDs) constitute the leading cause of death worldwide1.
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Vascular remodeling and stiffening are hallmarks of CVDs and primarily contribute to
CVD severity2. Dysregulated balance between collagen deposition and degradation has
been reported to promote vascular stiffening. Recently, we and others have shown that
activated vascular fibroblasts promote collagen biosynthesis in CVDs3–5. For instance, in
pulmonary hypertension (PH), a deadly enigmatic disease of the pulmonary vasculature, as
well as in a particularly deadly subtype pulmonary arterial hypertension (PAH), hormonal,
inflammatory, and environmental stresses such as hypoxia/ischemia, or mechanical cues

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activate resident adventitial fibroblasts at early stages of disease3. Such activation is

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characterized by increases in cellular proliferation in the expression of contractile proteins

as well as in the secretion of extracellular matrix (ECM) proteins, chemokines, cytokines,
growth, and angiogenic factors -- all processes that require energy. However, beyond
glycolysis, the metabolic requirement of activated pulmonary artery adventitial fibroblasts
(PAAF) remains unknown5. Thus, deciphering the metabolic circuits of PAAF activation
will offer much needed insight into the molecular underpinnings of this disease and provide
diagnostic and therapeutic entry points for identifying and treating disease more effectively.

Under the control of adventitial fibroblasts are the hundreds of ECM proteins and fibrillar
collagens that have been shown to provide the scaffold and give mechanical properties
to the vascular wall structure6–8. Dysregulation of the mechanical properties of vascular
cells microenvironment is increasingly recognized as a driver of aberrant vascular cell
reprogramming. As such changes in vascular collagen metabolism is capable of directly
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affecting resident vascular cell wall behaviors in a manner that influences overall vascular
tone and wall structure. The amino acid composition of collagen is atypical for proteins.
The most common motifs in the amino acid sequence of collagen are glycine-proline-X
and glycine-X-hydroxyproline, where X is any amino acid other than glycine, proline or
hydroxyproline. As such, around 33% of collagen amino acids are glycine, while proline
and hydroxyproline account for 20% of collagen amino acids9. Consequently, the massive
collagen secretion by activated fibroblasts observed during PH progression must require
these cells to adapt their metabolism to match their energetic needs.

The dynamic metabolism of vascular adventitial fibroblasts suggests an important link

between these cells and vascular stiffening with diet and nutrition. The central role of diet in
cardiovascular health and disease is well documented10. For instance, both population-level
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and wet bench research have connected obesity and dietary consumption with increased
CVD risk, while a low carbohydrate, low-fat diet promotes reduced CVD incidence11.
Emerging evidence has emphasized the importance of timed and intermittent dietary
ingestion in influencing global metabolic health in CVD12,13. Yet, the specific dietary
components that control vascular stiffening and downstream cardiopulmonary vascular
phenotypes in health and disease are poorly defined. This is particularly true in pulmonary
vascular diseases, where the impact of diet on risk and severity of PH has not been studied in
depth at a molecular level14.

Here, using metabolomic discovery platforms across primary diseased vascular cells, in vivo
rodent models of PH, and lung tissue of patient with PH, we sought to define the precise
metabolic requirements of pulmonary adventitial fibroblast (PAAF) during pathogenic
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activation. In doing so, we developed both targeted pharmacologic and dietary interventions
on those metabolic circuits that reduced collagen production and vascular stiffening, thus
improving vascular function in PH in vivo.

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RESULTS
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PAAF activation rewires glutamine and serine metabolism to sustain proline and glycine
biosynthesis.
To determine whether activation of PAAFs rewires cellular metabolism to sustain ECM
production, we performed a series of metabolomic studies on PAAF culture on soft or
stiff matrices and exposed to PH triggers. Here, a soft matrix was defined by a Young’s
(elastic) modulus of 1 kPa, stiff matrix was defined as 12 kPa consistent with our prior
studies of the stiffness of non-diseased and diseased pulmonary arterioles in rodents3,15.
Multiple PH triggers, including exposure to interleukin-6 (IL-6) and transforming growth
factor beta (TGF-β), matrix stiffening (12kPa), and hypoxia, activated PAAFs, as reflected
by increased contractility (increased myosin light chain 2 phosphorylation, P-MLC2; Figure
S1A–C). Steady-state metabolic cell profiles of activated PAAFs (Figure S1D–F) or PAAFs
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from PH donors (Table S1; Figure 1A and S1G) demonstrated that activation promoted
substantial global alterations of cell metabolism. Consistent with the notion that collagen
production by fibroblasts requires proline and glycine anabolism, pathway analysis revealed
that fibroblast activation significantly altered the steady-state levels of metabolites highly
enriched in pathways related to proline and glycine metabolism (Figures 1B and S1E).
Consistent with these results, the expression levels of several genes involved in proline and
serine biosynthesis were upregulated in activated PAAFs (Figure S1H–I). To determine any
preferential carbon sources for proline and glycine biosynthesis, we sought for metabolites
that are increasingly taken up by activated fibroblasts or diseased lungs. Steady-state
metabolomic analysis of conditioned media from either PH-PAAFs (Figure 1C) or activated-
PAAFs (Figure S1J) revealed a reduction of glutamine, serine, glucose (and, to lesser
extents, proline and glutamate), suggesting that these cells exhibit increased uptake of these
amino acids. Correspondingly, metabolic analysis of the trans-pulmonary plasma gradient
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of monocrotaline-treated rats showed that diseased lungs increased their consumption of

glutamine, serine, and glucose (Figures 1D–E). Similar results were obtained in the plasma
of patients with PH (Table S2) comparing amino acid levels between right atrial and
pulmonary capillary wedge positions (Figure 1F–G).

To discriminate between carbon sources (Figure 2A) that are taken up by cells from the
media and to determine if at least a portion of the metabolic rewiring observed in activated
PAAFs provides carbons for proline and glycine biosynthesis, we employed stable isotope
tracing studies, using [U-13C]-glucose, [U-13C]-glutamine, [U-13C]-glutamate, or [U-13C]-
serine (Figures 2B–E and S2). Cells were incubated with labeled metabolite for 4 hours.
These tracing experiments demonstrated that glucose, glutamine, and serine catabolism
were increased upon PAAF activation. Moreover, both glutamine-derived proline (M+5) and
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serine-derived glycine (M+3) levels increased in response to PAAF activation (Figures S2B
and S2D). In contrast, glucose and glutamate did not significantly account for proline and
glycine biosynthesis (Figures S2A and S2C). To derive further insight into the metabolic
rewiring of activated fibroblasts in vivo, we performed complementary experiments
quantifying glutamine and serine uptake. 18F-labeled fluoroglutamine (18F-FGln)16 coupled
with positron emission tomography (PET) scanning correspondingly revealed increased
glutamine absorption by diseased lungs and lung vasculature in monocrotaline-exposed rats

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with experimental PH (Figure 2F–H). To complement such PET imaging of glutamine

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uptake at tissue-scale resolution with glutamine and serine imaging at cellular resolution,
we leveraged a multi-isotope imaging mass spectrometry (MIMS), merging in vivo stable
isotope tracer methodology with nanoscale secondary ion mass spectrometry (Figure 2I–M).
Consistent with the in vitro tracing studies and 18F-FGln PET imaging, significantly elevated
glutamine and serine uptake was observed in the adventitia of diseased pulmonary arterioles
in monocrotaline-exposed PH rats. Together, our results indicate that activated-PAAFs
increase their glutamine and serine uptake, with these amino acids serving as substrates
for production of proline and glycine and consequent collagen biosynthesis and vascular
stiffening in PH.

Metabolic rewiring of activated fibroblast promotes the building of a diseased vascular

niche.
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To investigate whether glutamine and serine catabolism are required for vascular collagen
biosynthesis, we tested whether inhibition of glutaminase (GLS1) (the enzyme that converts
glutamine to glutamate, the first step of glutamine conversion to proline) and/or the serine
hydroxymethyltransferase (SHMT1) (the enzyme that converts the serine to glycine and
is the key isoform as compared to SHMT2 that is responsive to PH triggers, Figure
S1) controls collagen production by activated PAAFs. GLS1 inhibition, achieved via
siRNA (Figure S3A–C) or the small molecule CB-839 (Figure S3D–E), reduced proline
biosynthesis, while SHMT1 inhibition achieved via siRNA or the small molecule SHIN1
blunted glycine biosynthesis. Inhibition of both GLS1 and SHMT1 drastically rewired the
metabolism of activated PAAFs and diseased PAAFs and decreased both proline and glycine
biosynthesis (Figure S3A–E). Consistent with our hypothesis, siRNA knockdown of GLS1
or SHMT1, and to a greater extent siRNA knockdown of GLS1 and SHMT1, decreased
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collagen production by PAAFs activated by PH triggers such as IL-6 (Figure 3A), stiffness
(Figure S3F) or hypoxia (Figure S3G). Supplementation with permeant α-ketoglutarate
rescues collagen production (Figure S3H). Similar results were obtained in PH-PAAFs
treated with CB-839 and SHIN1 (Figure 3B and S3I).

Perivascular collagen remodeling by vascular fibroblast promotes a pro-proliferative

vascular niche, thus activating vascular cell proliferation in CVD. To investigate whether
glutamine and serine catabolism are required for these pathogenic processes, we leveraged
a matrix-derived cell culture platform. PAAFs were transfected with siRNAs for GLS1
and SHMT1 and allowed to produce and remodel ECM for five days. Then, fibroblasts
were removed, and the ECM was either analyzed for the quantity and quality of their
fibrillar collagen or used to plate naïve vascular cells (Figure 3C). siRNA knockdown
of GLS1 or SHMT1, and to a greater extent siRNA knockdown of GLS1 and SHMT1,
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decreased fibrillar collagen deposition and assembly, thereby decreasing vascular cells
proliferation (Figure 3D–E). Similar results were observed in PH-PAAFs treated with GLS1
and SHMT1 pharmacologic inhibitors (Figure 3F–G). Next, to determine whether the PAEC
and PASMC proliferation observed rely on the mechanical qualities of the collagen-rich
environment or the molecular content we performed a series of complementary experiments.
First, we analyzed the proliferation of vascular cells cultivated on soft or stiff matrices
and supplemented with or without proline and glycine. While matrix stiffening increased

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the proliferation of both PAEC and PASMC, proline/glycine supplementation did not
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affect cell proliferation (Figure S3J). Second, we investigated whether proline and glycine
supplementation can increase the proliferation rate of PAEC or PASMC cultivated on a
matrix from non-activated PAAFs (control-PAAFs). Consistent with our results showing
that proline and glycine did not increase the proliferation of cells cultivated on soft matrix,
supplementation with proline/glycine did not increase the proliferation of vascular cells
(Figure 3I). Together, these results offer evidence against a predominant role for the ECM
molecular content of these specific amino acids in controlling vascular cell proliferation. To
demonstrate the role of matrix stiffness in vascular cell proliferation, we cultivated vascular
cells on matrix produced by IL6-activated fibroblast and treated with or without BAPN, an
inhibitor of lysyl-oxidases (LOXs), the enzymes that crosslink the collagen. Consistent with
previous reports3,15,17, BAPN treatment reduced the matrix stiffness as measured by AFM,
(Figure 3H) and decreased the proliferation of vascular cells plated on top of the matrices
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(Figure 3I). Together, our results define glutamine and serine catabolism as crucial metabolic
processes, essential for fibroblast-driven vascular stiffening and remodeling.

YAP and TAZ are overarching regulators of vascular remodeling by diseased PAAFs.
To delineate the molecular mechanism controlling vascular remodeling by PAAFs during
PH progression, we sought to identify the overarching molecular regulator(s) of glutamine
and serine catabolism in activated fibroblasts. Recently, we and others have reported the
ability of the co-transcriptional factors YAP and TAZ to control both cell activities and
cell metabolism and thus adjust the metabolic needs of cells with their activities15,18,19.
Furthermore, several studies have reported the involvement of YAP and TAZ in CVD20,21
and fibroblast activation3,22. Therefore, to test whether YAP and TAZ globally control
fibroblast metabolism and especially glutamine/proline and serine/glycine metabolism, we
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analyzed the reported binding sites of TEADs (the transcriptional factors to which YAP
and TAZ bind to mediate their transcriptional effect23,24) as determined by chromatin
immunoprecipitation and DNA sequencing25,26 (ChIP-seq). Pathway enrichment analysis
revealed that, beyond cell proliferation and DNA/RNA metabolism, TEADs control a
large set of genes involved in cell metabolism and included genes involved in glutamine/
proline and serine/glycine metabolism (Figure 4A). To gain further insights on the role of
YAP/TAZ in the coordination of collagen production and the metabolic rewiring required
for collagen biosynthesis, we searched for TEAD binding sites in promoters of genes
involved in the metabolism-dependent circuit of collagen biosynthesis (Figure 4B–C).
TEAD ChIP seq analysis revealed that TEADs bind upstream to many genes from the
glutamine-to-proline and serine-to-glycine metabolic pathways (Figure 4C). To confirm
these results, we performed YAP ChIP-qPCR experiments in PH-PAAFs (Figure 4D). We
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demonstrated that YAP binds the promoter region of a large set of genes involved in
glutamine and serine rewiring as well as genes involved in collagen metabolism (Figure 4D).
Moreover, manipulating YAP and TAZ expression (Figure S4A) modulated these genes at
the transcriptional level (Figure S4B) and at the protein level for GLS1 and SHMT1 (Figure
S4A). Together, these results demonstrate that YAP and TAZ coordinate the transcription
of a large pool of genes involved in glutamine to proline and serine to glycine metabolism,
as well as collagen metabolism, indicating that YAP and TAZ may orchestrate collagen
biosynthesis via control of amino acid metabolism.

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To investigate more precisely whether YAP and TAZ metabolically control collagen
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biosynthesis, we analyzed the steady-state metabolic profile of YAP and TAZ knockout
cells (Figures 4E–F and S5A–B). Modulating YAP and TAZ expression in activated PAAFs
promoted a substantial global alteration of cell metabolism. Beyond pathways related to
glucose metabolism, the analysis revealed that siRNA knockdown of YAP and TAZ in
activated-PAAFs modulated the steady-state levels of metabolites highly enriched in proline
and glycine metabolism pathways (Figures 4F and S4B). Consistent with the role of YAP
and TAZ in the control of the glutamine to proline and glycine to serine metabolism,
siRNA knockdown of YAP and TAZ in activated-PAAFs decreased the glutamine-derived
proline (M+5) and the serine-derived glycine (M+3) levels (Figures 4G–H and S5C–F) and
decreased collagen production (Figures 4I and S4C–D). Furthermore, we demonstrated that
manipulating YAP and TAZ expression in activated fibroblasts decreased fibrillar collagen
deposition and assembly in the ECM (Figures 4J–L and S4E), thereby decreasing the
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proliferation of naive vascular cells (Figures 4J–L and S4E). Together, these results showed
that YAP and TAZ directly control the rewiring of glutamine and serine into proline and
glycine metabolism to sustain collagen biosynthesis.

Next, we investigated whether YAP and TAZ metabolically control collagen biosynthesis
in vascular fibroblast in vivo to build a diseased vascular niche promoting PH. Specific
conditional knockdown of YAP and TAZ in fibroblasts of pulmonary-specific IL-6
transgenic mice (Figure 5A) – an inflammatory mice model of PH27—decreased GLS1
(Figure 5B) and SHMT1 (Figure 5C) expression in the pulmonary arteries, decreased the
pulmonary vascular collagen content (total and fibrillar; Figure 5D–F), thereby decreasing
pulmonary arterial stiffness as measured by atomic force microscopy (AFM; Figure 5G).
Given the alterations occurring in the pulmonary vascular ECM, the deletion of YAP and
TAZ in fibroblasts decreased vascular mural cell proliferation (Figure 5H) and improved
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downstream histologic (Figure 5I), hemodynamic (Figure 5J), and right ventricular indices
(Figure 5K) of PH. Similar results were observed in a second rodent model of PH where
PH is induced by chronic exposure to hypoxia (Figure S6). Together, our results demonstrate
that YAP and TAZ coordinate the metabolic needs of activated vascular fibroblasts with their
collagen remodeling activities to build a diseased vascular niche that favors vascular mural
cell proliferation.

Targeting proline and glycine anabolism decreases collagen production and vascular
stiffening, thus improving PH in vivo.
We next investigated whether targeting glutamine and serine catabolism is sufficient to
decrease vascular stiffening and improve pulmonary vascular function in a pre-clinical
model of PH. Monocrotaline-exposed rats were treated with either CB-839 or SHIN1 or the
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combination of inhibitors (Figure 6A). Pharmacologic inhibition of both GLS1 and SHMT1
decreased the pulmonary vascular collagen content (Figure 6B–C), decreased the pulmonary
arterial stiffness (Figure 6D), thus decreasing vascular cell proliferation (Figure 6E). As a
result, GLS1 and SHMT1 inhibition significantly decreased pulmonary arteriolar remodeling
and muscularization (Figure 6F), right ventricular systolic pressure (Figure 6G), and right
ventricular remodeling (Figure 6H). In comparison, while CB-839 alone was effective in
reducing vascular collagen content, vascular stiffness, proliferation as well as histologic and

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hemodynamic parameters of PH, SHIN1 alone was only modestly active alone across these
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parameters. Notably, SHIN1 decreased the total amount of fibrillar collagen in the lungs but
did not modulate the soluble/insoluble ratio (Figure 6B). Together, these drugs demonstrated
a more robust improvement than either drug alone, particularly across collagen quality,
stiffness, proliferation, and muscularization.

Based on these results demonstrating the glutamine and serine metabolic requirement for
collagen biosynthesis in vivo during PH development, we investigated whether decreasing
glutamine and serine bio-availability via dietary intervention (Figure 7A) could reduce
collagen biosynthesis and thus improve cardiopulmonary function. We first studied whether
decreasing glutamine and serine ingestion chronically reduced glutamine and serine
bioavailability (Figure 7B). Indeed, we found rapid and sustained decreases in circulating
levels of glutamine and serine in the plasma of rats fed with glutamine- and serine-free
diets, while displaying minimal or inconsistent impact on glucose (Figure 7B), Second, to
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assess the effect of dietary serine and glycine (SG) restriction in a pre-clinical PH model,
we transferred 12-week old rats from a normal chow diet to experimental diets (synthetic
diet, Ctrl; or Gln−/ser− synthetic diet) 18 days before monocrotaline injection. Three weeks
later, we analyzed for alterations of amino acid metabolism, vascular stiffening, and PH.
Transferring rats from normal chow diet to an experimental glutamine- and serine-free diet
significantly reduced plasma levels of glutamine and serine (Figure 7C), pulmonary vascular
collagen content (Figure 7D–E), pulmonary arterial stiffness (Figure 7F), and vascular cell
proliferation (Figure 7G–H). Consequently, such an amino-acid restricted diet significantly
decreased pulmonary arteriolar remodeling and muscularization (Figure 7I), right ventricular
systolic pressure (Figure 7J), and right ventricular remodeling (Figure 7K). Together, our
results, offer foundational support for dietary intervention targeting glutamine and serine to
improve pulmonary vascular stiffening, remodeling, and downstream PH.
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DISCUSSION
Our findings delineate a YAP/TAZ-dependent metabolic circuit controlling collagen
biosynthesis by activated vascular fibroblasts. Specifically, our results establish glutamine
and serine catabolism as crucial steps for collagen biosynthesis. These results carry broad
implications for our fundamental understanding of collagen metabolism - a dysregulated
process in several diseases such as cancer and across CVDs such as PH. Furthermore, our
study reveals a much broader and more complex regulation of the extracellular vascular
niche that could be metabolically targeted. As such, our findings provide scientific guidance
for incorporating pharmacologic and dietary interventions as amino acid-based therapies in
these deadly diseases.
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Interplay among the transcriptional coactivators YAP and TAZ with cell metabolism has
recently gained traction19,28. YAP and TAZ are activated by various stimuli inherent to the
Hippo signaling pathway, glucose metabolism, as well as mechanical signals. YAP/TAZ
control cell growth, stem cell maintenance, and tissue homeostasis23. Given the energy
requirement of cell proliferation and survival, YAP and TAZ are inhibited when the energy
level is low29–31. Conversely, YAP and TAZ control several metabolic pathways, including
glutaminolysis and anaplerosis, to sustain the metabolic needs of cellular proliferation15,18.

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Moreover, YAP and TAZ are activated during tissue repair processes and sustain fibroblast
activation in fibrosis21,22. Consistent with the role of YAP/TAZ and the Hippo signaling
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pathway in PH3,32, our findings highlight a much more complex regulation of metabolism
by YAP and TAZ in tissue fibrosis – whereby both glutamine and serine catabolism
conspire to generate proline and glycine that ultimately are crucial for de novo synthesis
of collagen. It remains to be seen if glutamine and serine pathways can also act in
a reciprocal feedback loop to further activate YAP/TAZ and reinforce this metabolic
reprogramming in activated PAAFs. Recent data have shown that extracellular uptake rather
than synthesis of proline may be a primary mechanism by which pulmonary vascular
endothelium can generate collagen33. Our data indicate that activated PAAFs – cell types
that carry the bulk of responsibility for collagen deposition – rely more prominently on
proline biosynthesis for new collagen synthesis and vascular stiffening. Given the increasing
appreciation of molecular communication and interdependence between the endothelium
and adventitium34,35, an intriguing mechanism could include a process by which proline
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taken up by diseased endothelium ultimately originates from proline synthesized from

glutamine in fibroblasts. Furthermore, given the key roles of endothelial-to-mesenchymal
transition and smooth muscle transition between contractile and synthetic states in PH
pathogenesis, it will be important to determine whether dynamic changes in glutamine
and serine metabolism (observed across the vessel wall, can contribute to these or other
phenotypic transitions of vascular cells. Development of approaches allowing metabolic
assessment at single-cell level such as MIMS coupled to spatial transcriptomics may be
necessary to fully decipher such putative metabolic interactions.

Beyond PH, these findings may carry important implications for diseases where ECM
deposition and/or remodeling by tissue-resident fibroblasts drive pathogenesis, such as
peripheral vascular diseases, heart failure with preserved ejection fraction (HFpEF), cancer,
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and tissue fibrosis (i.e., lung, liver, kidney), in general. Future work is warranted to
define whether non-vascular and vascular fibroblasts in other anatomic compartments
also rely predominantly on glutamine and serine catabolism for collagen synthesis. In
particular, intracellular glutamine content may serve as a central rheostat of diverse cellular
function – one that is even more versatile than currently characterized. For example, recent
findings have emerged regarding the mechano-induced glutamylation and stabilization of
microtubules that can drive cancer cell migration36. Since glutamine levels define a limiting
metabolic requirement for collagen synthesis and fibrosis, an intriguing hypothesis now
may emerge whereby shunting of glutamine to microtubule glutamylation vs. to collagen
deposition may either facilitate metastatic cancer invasion or more dense tissue fibrosis. In
vascular diseases beyond PH, the balance of such fundamental processes may otherwise
define the extent of angiogenesis vs. scar or infarct size across a variety of vascularized
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tissues experiencing ischemic or hypoxic insults.

Beyond glutamine metabolism, our study emphasized the YAP/TAZ-dependent control over
serine/glycine metabolism. Although often associated with glucose catabolism, here we
demonstrate that glycine biosynthesis relies on exogenous serine catabolism. Whereas non-
activated cells exhibit lower demands for serine and glycine, hyperproliferative cancer cells
demonstrate hyperactive intracellular serine and glycine synthesis and are addicted to de
novo production37,38. Our findings demonstrate that similar metabolic processes are at play

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 Rachedi et al. Page 11

in diseased vasculature, furthering the notion of parallel features of cancer and peripheral
and pulmonary vascular disease39,40. Namely, our findings implicate SHMT1 as a crucial
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mediator of PH pathogenesis, similar to our understanding in cancer38.

In identifying the crucial metabolic pathways required for collagen biosynthesis in fibrotic
disease, our work offers the potential for non-invasive imaging tools for diagnosis and
prognosis in PH. Despite its morbid prognosis, PH, and particularly PAH, is often
misdiagnosed or ignored, with an average time of >40 months between onset of symptoms
to diagnosis and progression of PAH severity41. A crucial need exists to develop non-
invasive diagnostic imaging tools, particularly that can detect early disease stages and reflect
molecular alterations that drive disease in the lung, vasculature, and right ventricle (RV)42.
While our use of MIMS offered research applicability, this technique is too specialized,
time-consuming, and low-throughput to offer a viable diagnostic platform for widespread
clinical use. In contrast, based on our advanced insights into glutamine metabolism in
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PH, positron emission tomography (PET) may offer a clinically available platform for
imaging changes in the metabolic state of the vasculature and right ventricle in PH. PET
has been applied in PAH to image 18F-fluorodeoxyglucose (18FDG) uptake as a reflection
of increased glycolysis42 particularly in the RV43. However, this specific PET tracer in
PAH is limited by inconsistent results42 and inability to detect early stages of disease.
Beyond 18FDG, highly glutaminolytic tissue are avid for glutamine, as shown by its uptake
in cancers44, and this process is quantifiable by PET. 18F-(2S,4R)-4-fluoroglutamine (18F-
FGln) is a single stereoisomer analog of glutamine with promising results for imaging of
tumors45 such as gliomas16 and breast cancer44. When coupled with our published findings
that glutamine levels decrease in the pulmonary circulation in human patients with PAH15,
our preclinical data with this tracer strengthen the notion that 18F-FGln and glutamine
uptake could be a feasible modality to track in patients with PAH. Given the dearth of
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effective imaging tests for PAH and PH in general, a first-in-human trial of 18F-FGln PET in
PH could offer a translational avenue to pursue glutamine imaging to define disease severity
and risk, including at early disease time points. 18F-FGln PET could offer a clinical trial
outcome surrogate, reflecting the molecular pathogenesis of PH. It could also aid in clinical
trial recruitment of “super-responder” patients with PH, thus decreasing patient numbers for
recruitment and potentiating an era of precision medicine for PH.

Coupled with furthering such non-invasive platforms of identifying patients with PH avid
for glutamine uptake, our findings also offer a roadmap toward more effective and precise
pharmacologic therapies for PH. Previously we have shown that glutaminolysis supports
PAEC and PASMC proliferation, migration, and contractility via anaplerosis. By now
identifying glutamine metabolism as key step for collagen biosynthesis in PAAF, our
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findings highlight the pleiotropic activity of glutamine across the pulmonary vasculature.
It remains unclear if serine metabolism carries as many roles, perhaps serving as an
explanation as to why targeting GLS1 appears consistently more effective than targeting
serine metabolism alone. Yet, the interconnected nature of these metabolic pathways opens
up the possibility of combinatorial therapies. Namely, while isolated inhibition of GLS1
can improve PAH15, the synergism of inhibiting both YAP1 and GLS1 in PH has been
described46. We now further define the therapeutic benefit of simultaneous delivery of GLS1
and SHMT1 inhibitors in the diseased pulmonary vasculature. These findings set the stage

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 Rachedi et al. Page 12

for the clinical development of combination of such synergistic agents for more robust
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and metabolic control of PH manifestations. However, dosing strategies would be complex

and likely would necessitate more targeted administration. Given the strong track record
of poly(lactic-co-glycolytic) acid (PLGA) microparticles for inhalation47 including in PH48
recently, an inhaled microparticle for local, controlled release of these metabolic therapies
may be appealing.

Finally, by defining the limiting carbon sources for collagen biosynthesis, our study reveals
a unique opportunity to introduce dietary modulation in the management of PH and CVDs,
in general. It is well known that dietary changes play a dominant role49,50 in a number of
CVDs. Beyond CVDs, dietary intervention has been proposed to ameliorate tissue fibrosis,
such as in fatty liver diseases51. Moreover, altering the timing of daily food ingestion in
the form of intermittent fasting has increasingly been identified as an effective modulator
of cellular and physiologic metabolic parameters, with robust implications on aging and a
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variety of chronic diseases52. More recently, intermittent fasting has been proposed as a
potential intervention for right ventricular dysfunction in PAH14. However, a diet aimed at
modulating specific ingested amino acid content has not been tested to our knowledge as
a precise therapeutic intervention for fibrosis or cardiopulmonary diseases. Although both
glutamine and serine (as well as proline and glycine) can be synthesized from sources
other than the diet, our preclinical data indicate that targeted dietary restriction of glutamine
and serine are effective in reducing these amino acid levels in lung tissue, suggesting a
decrease of their overall bioavailability. Future studies are warranted to determine whether
dietary sources of these amino acids predominantly and directly contribute to lung vascular
fibrosis in PH or whether dietary alterations may also serve as a primary but indirect trigger
to altered specific amino acid production and/or degradation in lung as PH progresses.
Furthermore, it is possible that genetic variability in the enzymes that transport and
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metabolize these dietary amino acids in the gut may influence the therapeutic effects of
such dietary restriction. Translating this approach to human patients would also necessitate
greater insight into the complex cooking and processing that could alter the amino acid
composition of any prescribed dietary prescription. Progress toward network medicine53 and
artificial intelligence could help us to systematically define the complex link between the
diet with the genome and disease and thus prepare for feasible dietary interventions in PH
and beyond.

In sum, we identify the precise metabolic and amino acid requirements for vascular
fibroblast activity. This study provides a fundamental insight into the regulation of collagen
metabolism and fibrosis, which is critical for managing PH and holds relevance for other
fibrotic diseases across other CVDs and beyond. Moreover, our study offers the potential for
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metabolic PET imaging platforms for more effective non-invasive diagnostic and prognostic
testing in PH and vascular stiffening. It also guides developing pharmacologic and dietary
therapies targeting metabolic crosstalk in the diseased vasculature.

Limitations of study:
Limitations to this study should be acknowledged. When interpreting transpulmonary
gradient analyses from rats and humans, we acknowledge that streaming from hepatic veins

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 Rachedi et al. Page 13

may confound collection from either the inferior vena cava (rat) or right atria (human). To
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demonstrate the role of YAP and TAZ in vascular fibroblasts in vivo, a cell type-enriched
deletion of YAP and TAZ was accomplished using mice expressing the Cre transgene
driven by a tamoxifen-inducible Col1a2 promoter. Although Col1a2 is primarily expressed
in fibroblasts54, smooth muscle cells also express this gene. Thus, we cannot rule out that
at least a portion of the downstream phenotype may be driven by smooth muscle deletion
of YAP and TAZ and that cell type’s contribution to collagen deposition. Our experiments
with dietary restriction also do not account for the kinetics of endogenously produced
and non-diet administered amino acids, which could confound an interpretation of direct
cause-effect in diet restriction with PH. More broadly, our study is not set up to define
the dynamic and likely bidirectional contribution of total metabolite content across the
plasma, lung vasculature, and other tissue or dietary sources. To address this point, future
metabolomic studies of the lung in PAH could be envisioned that are designed similarly to
prior work quantifying fuel use in the failing human heart55.
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STAR METHODS
RESOURCE AVAILABILITY
Lead contact—Further information and requests for resources and reagents should
be directed to and will be fulfilled by the Lead Contact, Thomas Bertero, PhD
(thomas.bertero@univ-cotedazur.fr).

Materials Availability—All unique/stable reagents generated in this study are available

from the Lead Contact without restriction.

Data and Code Availability

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• All of the data supporting this study are included in the article. All raw data used
to generate the figures throughout the manuscript can be found within the Data
S1 document

• This paper does not report original code

• Any additional information required to reanalyze the data reported in this paper
is available from the lead contact upon request.

EXPERIMENTAL MODELS AND SUBJECT DETAILS

Cell culture—Primary human pulmonary arterial adventitial fibroblasts (PAAF) were
purchased from Sciencell. Cells used in this study were within 7 passages and were cultured
(37°C, 5% CO2) in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco) supplemented
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with 10% fetal bovine serum (Gibco), Glutamine (2mM, Gibco) and Penicillin/Streptomycin
(1%, Gibco). A triple gas incubator set at 1% oxygen, 94% nitrogen, and 5% carbon dioxide
was used for hypoxic conditions. PAAFs from patients with PH (PH-PAAF) were isolated
from explanted lungs at Marie Lannelongue Hospital. To isolate PAAFs, vascular cells
from dissected pulmonary arteries were cultured in DMEM supplemented with 0.5% FCS.
In this condition, PH-PAAFs that outgrew PAEC and PASMC in culture were isolated
by differential trypsinization. These cells were cultured in DMEM supplemented with

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10% FCS, 2mM glutamine, 1% Pen/Strep and insulin-transferrin-selenium (Invitrogen).

Constitutive activation of PH-PAAFs was verified by measuring Vimentin and α-SMA
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expression as well as by their ability to contract a collagen I rich gel in the absence of
serum. PH-PAAFs from at least five different patients were randomly used throughout
the experiments. All cells were grown in collagen-coated plastic (50 μg/mL) at 37°C in
a humidified 5% CO2 atmosphere. Experiments were performed at passages 2–10. All
cells were routinely tested for mycoplasma by PCR and were carefully maintained in a
centralized cell bank. Primary human (Lonza) pulmonary arterial endothelial cells (PAECs)
were grown in EBM-2 basal medium supplemented with EGM-2 MV BulletKit (Lonza).
Primary human (Lonza) pulmonary arterial smooth muscle cells (PASMCs) were grown in
SmGM-2 (Lonza) For the studies dependent on matrix stiffness, collagen-coated hydrogel
pre-plated in culture wells (Matrigen) was generated from a mix of acrylamide and bis-
acrylamide coated with collagen. Cells were cultured, passaged, and harvested while on top
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of the hydrogel, using standard cell culture techniques.

Human studies—Patients with PAH from whom PAAFs were isolated were part of the
French Network on Pulmonary Hypertension, a program approved by our institutional
Ethics Committee, and gave written informed consent (Protocol N8CO-08-003, ID RCB:
2008-A00485-50, approved on June 18, 2008). Patients with PAH and controls from whom
plasma was obtained were part of a University of Pittsburgh School of Medicine cohort.
All patients gave written informed consent. The protocol was approved by the institutional
review board at the University of Pittsburgh (IRB: STUDY19050364).

In vivo animal studies

Mouse strains.: Col1a2-Cre-ER, Yap1fl/fl/Tazfl/fl; and IL-6 transgenic mice (C57/Bl6
background) have been previously described27 or imported from the NCI mouse repository
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and the Jackson Laboratories.

Rodent models of PH
• Inducible cell-specific YAP and TAZ knockout mice. Col1a2-Cre-ER mice were
crossed onto Yap1fl/fl/Tazfl/fl mice. Tamoxifen was diluted at 20 mg/ml in
corn oil (Sigma). Three daily intraperitoneal injection (IP) doses of 30 mg/kg
of tamoxifen were administered to ten-week-old mice (C57Bl6) in order to
knockout YAP and TAZ in cells expressing Col1a2 – a marker enriched in
fibroblasts. Two weeks after injection, exposure to hypoxia commenced.

• Hypoxia-induced PH. Mice were exposed to normobaric hypoxia (10% O2;

OxyCycler chamber, Biospherix Ltd, Redfield, NY) or normoxia (21% O2)
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for 3 weeks. In all cohorts, on day 21 after hypoxia exposure, right heart
catheterization was performed followed by harvesting of lung tissue, paraffin
embedding, or cryopreservation with OCT (Sigma-Aldrich).

• Monocrotaline-induced PH. Male Sprague-Dawley rats (10–14 week old) were

injected with 60 mg/kg monocrotaline at time 0. At 3 weeks’ post-exposure, right
heart catheterization was performed followed by harvest of lung tissue, paraffin
embedding, or cryopreservation with OCT.

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Trans-pulmonary plasma gradient of monocrotaline-treated rats.: Male Sprague-Dawley

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rats, aged 10–14 weeks, were divided into two groups: the PH rats, which received an
injection of 60 mg/kg monocrotaline, and the control rats, which received a saline injection.
After a three-week period, 200 μl of blood was collected from the left common carotid artery
and the inferior vena cava into EDTA tubes. The blood samples were then centrifuged, and
the resulting plasma was stored at −80°C for future use.

Inhibition of GLS1 and/or SHMT1 in PH rats.: To induce PH, male Sprague-Dawley rats
(10–14 weeks old) were injected i.p. with 60 mg/kg monocrotaline (Sigma-Aldrich). After 7
days, serial i.p. injections were given daily of CB-839 (10 mg/kg; Selleck Chemicals) and/or
SHIN1 (5mg/kg; Cayman chemical) versus vehicle control. In all cohorts, on day 21 after
monocrotaline injection, right heart catheterization was performed followed by harvesting of
lung tissue, paraffin embedding, or cryopreservation with OCT (Sigma-Aldrich).
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Diets.: On standard chow, dietary amino acids are derived from whole proteins contained
in the raw ingredients (wheat, wheatfeed, barley, de-hulled extracted toasted soya, maize
and fish meal), with a small amount of purified lysine added as a supplement. Two
sets of experimental diets were used, both based on Purified Amino Acid Diet from
SAFE Nutrition Service: control synthetic diet contained all essential amino acids plus
serine, glutamate, proline, and glycine; Glutamine and serine-free diet was the same as
synthetic diet-control, but without serine, glutamate, proline, and glycine, with the other
amino acid levels increased proportionally to achieve the same total amino acid content.
From weaning, rats received standard chow. Male Sprague-Dawley rats, aged 10–14
weeks, were assigned to three distinct groups: standard chow; synthetic control diet; or
glutamine and serine-free diet. After 18 days, Sprague-Dawley rats were injected i.p. with
60 mg/kg monocrotaline (Sigma-Aldrich). Right heart catheterization was conducted at day
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21 following monocrotaline injection in all experimental groups. Subsequently, lung tissue

was harvested and either paraffin embedded or cryopreserved using OCT (Sigma-Aldrich).

Human plasma harvesting: To collect blood from subjects from the main pulmonary
artery, clinically indicated right heart catheterization procedures were performed by standard
protocol via a right internal jugular approach under fluoroscopic guidance. The catheter was
positioned into the main pulmonary artery, as confirmed by fluoroscopy and hemodynamic
waveforms. Blood was drawn from the distal port and collected in vacutainer tubes with K+-
EDTA. Plasma was extracted after centrifugation of blood, followed by storage at −80°C.

METHOD DETAILS
Plasmids, antibodies, and reagents—The YAP1 coding sequence was purchased
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(Addgene; Plasmid #18881) and sub-cloned in the pCDH-CMV-MCS-EF1-copGFP (System

Biosciences) using EcoRI and NotI restriction sites. The lentiviral parent vector expressing
GFP was used as a control. Stable expression of these constructs in PAAFs was achieved by
lentiviral transduction. Constructs were verified by sequencing. CB-839 (S7655) and SHIN1
(Cat. No. 6998) were purchased from Selleckchem and Tocris respectively. The following
commercially available antibodies were used for western blotting and immunofluorescence:

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 Rachedi et al. Page 16

HRP-conjugated donkey anti-mouse IgG (715-035-150) and HRP-conjugated anti-rabbit

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IgG (711-035-152) were purchased from Jackson ImmunoResearch Laboratories.

siRNA and plasmid transfection—Cells were plated on collagen-coated plastic

(50μg/mL) and transfected 24h later at 70–80% confluence using siRNA (25nM)
and Lipofectamine 2000 reagent (Life Technologies), according to the manufacturers’
instructions. Eight hours after transfection, cells were trypsinized and re-plated on hydrogel.
Forty-eight after transfection cells were harvest for analysis. siRNA ON-TARGETplus
Human or non-targeting control siRNAs (D-001810-01) were purchased from Horizon
Discovery). siRNA ON-TARGETplus Human sequences are provided in the key resources
table.

Lentivirus production—HEK293T cells were transfected using Lipofectamine 2000

(Thermo Scientific) with lentiviral plasmids along with packaging plasmids (pPACK,
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System Biosciences), according to the manufacturer’s instructions. Virus was harvested,

sterile filtered (0.45 μm), and utilized for subsequent infection of PAAFs (24–48-hour
incubation) for gene transduction.

Immunoblot assays—Forty-eight hours after plaiting, cells were lysed in RIPA buffer
(Pierce) or directly in Laemmli’s buffer. After denaturation, protein lysates were resolved by
SDS-PAGE and transferred onto a PVDF membrane (Millipore). Membranes were blocked
with 2% BSA in TBS tween20 0.1% and incubated in the presence of the primary and
then secondary antibodies. After washing, immunoreactive bands were visualized with ECL
(Millipore) and analyzed on Fusion-FX Imager (Vilber).

Targeted LC-MS—Metabolite extraction was performed essentially as described18,36 with

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minor modifications. Briefly, metabolites were extracted from cultured cells on dry ice
using 80% aqueous methanol precooled at −80°C. Insoluble materials from both cell and
supernatant extractions was removed by centrifugation at 20,000 g for 15 minutes at
4°C. The supernatant was evaporated to dryness by SpeedVac at 42 °C, the pellet was
resuspended in LC-MS water, and metabolites were analyzed by LC-MS.

LC-MS analysis was performed on a Vanquish ultra-high performance liquid

chromatography system coupled to a Q Exactive mass spectrometer (Thermo) that was
equipped with an Ion Max source and HESI II probe. External mass calibration was
performed every seven days. Metabolites were separated using a ZIC-pHILIC stationary
phase (150 mm × 2.1 mm × 3.5 mm; Merck) with guard column. Mobile phase A
was 20 mM ammonium carbonate and 0.1% ammonium hydroxide. Mobile phase B was
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acetonitrile. The injection volume was 1 μL, the mobile phase flow rate was 100 μL/min, the
column compartment temperature was set at 25 °C, and the autosampler compartment was
set at 4 °C. The mobile phase gradient (%B) was 0 min, 80%; 5 min 80%; 30 min, 20%; 31
min, 80%; 42 min, 80%. The column effluent was introduced to the mass spectrometer with
the following ionization source settings: sheath gas 40, auxillary gas 15, sweep gas 1, spray
voltage +/− 3.0 kV, capillary temperature 275 °C, S-lens RF level 40, probe temperature 350
°C. The mass spectrometer was operated in polarity switching full scan mode from 70–1000
m/z. Resolution was set to 70,000 and the AGC target was 1×106 ions. Data were acquired

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 Rachedi et al. Page 17

and analysed using TraceFinder software (Thermo) with peak identifications based on an
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in-house library of authentic metabolite standards previously analysed utilizing this method.
For all metabolomic experiments, the quantity of the metabolite fraction analysed was
adjusted to the corresponding cell number calculated upon processing a parallel experiment.

Stable isotopic tracing analysis—To trace the effect of glucose or glutamine or

glutamate or serine on amino acid and glucose metabolism, PAAFs were grown 48h on
10 cm plate in DMEM containing 10% FBS, 20mM glucose, 2mM glutamine and then
transferred into glucose-free (with 2 mM glutamine) or glutamine free (with 20mM glucose)
or serine free (with 2 mM glutamine and 20mM glucose) DMEM containing 10% dialyzed
FBS and supplemented with either 20mM U-13C-glucose or 2mM U-13C-glutamine, or
5mM U-13C-glutamate, or 0.5mM U-13C-serine respectively for 4 hours prior to metabolite
collection. All media were removed, and plates were kept tilted for few seconds to allow any
additional media collection in corner of plate. Cells were immediately treated with 4 ml of
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80% ice-cold methanol (−80°C) and transferred to −80°C freezer for 20 minutes. Cell plates
were resuspended on dry ice and the collected lysate/methanol suspension was transferred
to 15 ml conical tubes kept on dry ice. Suspension was centrifuged at full speed (21,000
g) for 5 minutes at 4°C. Supernatants were then transferred to 50 ml conical tubes on dry
ice. The pellets were resuspended with 500 μl 80% methanol (−80°C) and a combination of
vortexing and pipetting up and down. The suspension was centrifuged at full speed (21,000
g) for 5 minutes at 4°C. Supernatant were transferred to 50 ml conical tubes on dry ice.
Pellets obtained after centrifugation were dissolved in urea 8 M (Tris 10 mM pH 8.0) at
60°C, to later measure protein concentration for normalization. After pooling the metabolite
extractions, the samples are completely dried under a nitrogen gas apparatus (N-EVAP) and
submitted for LC-MS analysis.
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Samples were analyzed by High-Performance Liquid Chromatography and High-Resolution

Mass Spectrometry and Tandem Mass Spectrometry (HPLC-MS/MS). Specifically, system
consisted of a Thermo Q-Exactive in line with an electrospray source and an Ultimate3000
(Thermo) series HPLC consisting of a binary pump, degasser, and auto-sampler outfitted
with a Xbridge Amide column (Waters; dimensions of 4.6 mm × 100 mm and a 3.5 μm
particle size). The mobile phase A contained 95% (vol/vol) water, 5% (vol/vol) acetonitrile,
20 mM ammonium hydroxide, 20 mM ammonium acetate, pH = 9.0; B was 100%
Acetonitrile. The gradient was as following: 0 min, 15% A; 2.5 min, 30% A; 7 min, 43%
A; 16 min, 62% A; 16.1–18 min, 75% A; 18–25 min, 15% A with a flow rate of 400
μL/min. The capillary of the ESI source was set to 275 °C, with sheath gas at 45 arbitrary
units, auxiliary gas at 5 arbitrary units and the spray voltage at 4.0 kV. In positive/negative
polarity switching mode, an m/z scan range from 70 to 850 was chosen and MS1 data
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was collected at a resolution of 70,000. The automatic gain control (AGC) target was set
at 1 × 106 and the maximum injection time was 200 ms. The top 5 precursor ions were
fragmented, in a data-dependent manner, using the higher energy collisional dissociation
(HCD) cell set to 30% normalized collision energy in MS2 at a resolution power of 17,500.
The sample volumes of 10 μl were injected. Data acquisition and analysis were performed
by Xcalibur 4.0 software and Tracefinder 2.1 software, respectively (both from Thermo
Fisher Scientific).

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4-18F-(2S,4R)-fluoroglutamine (18F-FGln) PET imaging—Synthesis of the precursor

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for 18F-FGln was performed as previously described56. Production of 4-18F-(2S,4R)-

fluoroglutamine (18F-FGln) was performed as previously described16. At 3 weeks’ post-
treatment, PBS- or monocrotaline-treated rats were anesthetized with 3% isoflurane and
injected with 1 mCi of 18F-FGln via the lateral tail vein. Immediately after injection,
PET data were collected during a 60-minute dynamic acquisition with a medium-low
magnification CT (full rotation, 220 projections, 80kV and 500 mA with a 550 ms exposure
and 2×2 binning) following the PET. PET data were reconstructed using a 2D filtered back-
projection (2DFBP) algorithm. CT data were reconstructed at a down sample of 2 with the
Siemens Feldkamp algorithm. Using Siemens Inveon Research Workplace software (IRW),
individual region of interest (ROI) slices were drawn across relevant tissue compartments
(left and right ventricles, lung space and muscle), and the slices for each compartments
were interpolated to give a volumetric ROI for that compartment. In the case of the lung
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space, the volumetric ROI was thresholded to give representations of both airspace and
bronchovasculature. The SUVmean was determined for each tissue compartment. The SUV
values in these regions were each normalized against the SUVmeanin muscle.

Multi-isotope Imaging Mass Spectrometry—Pulmonary vessels were analyzed with

a NanoSIMS 50L instrument (Cameca), an ion microscope which enables visualization
and quantification of stable isotope tagged metabolic tracers at subcellular resolution
along with histologic information. Incorporation of 15N-serine or 15N-glutamine, and 2H-
glutamine were derived from the simultaneous recording of 12C21H−, 12C14N−, 12C22H−,
and 12C15N− images using previously developed analytical approaches33,55. Ratio images,
12C15N−/12C14N− (a proxy for 15N/14N) and 12C 2H/12C 1H (a proxy for 2H/1H) were
2 2
analyzed using a custom plugin to ImageJ: OpenMIMS 3.0: https://github.com/BWHCNI/
OpenMIMS57. Isotope ratio data are shown as hue saturation intensity (HSI) images. The
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lower bound of the scale (blue) is set at natural background. The upper bound is set to
visually demonstrate differential labeling. Scaling changes affect the color pattern; however,
the underlying quantitative isotopic ratio data is unaffected. 15N-glutamine image acquisition
(Figure 2I–J) was performed with ‘mosaic imaging.’ Alternatively, targeted vessel imaging
was pursued in Figure 2K–L. Specifically, vessels were identified based on size, the
coordinates were recorded, and higher resolution images were then acquired by directly
analyzing individual vessels without capturing surrounding lung parenchyma. This was done
to minimize analytical ‘dead space.’

Messenger RNA extraction—Cells were homogenized in 1 mL of QiaZol reagent

(QIAGEN). Total RNA content was extracted using the miRNeasy kit (QIAGEN) according
to the manufacturer’s instructions. Total RNA concentration was determined using a
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ND-1000 micro-spectrophotometer (NanoDrop Technologies).

Quantitative RT-PCR of messenger RNAs—Messenger RNAs were reverse

transcribed using the Multiscript RT kit (Life Technologies) to generate cDNA. cDNA
was amplified via fluorescently labeled Taqman primer sets using an Applied Biosystems
7900HT Fast Real Time PCR device. Fold-change of RNA species was calculated using the
formula (2-ΔΔCt), normalized to RPLP0 expression.

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ChIP-qPCR—PH-PAAF were cultivated on plastic for 48h. Cells were dual cross-linked
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with 2 mM disuccinimidylglutarate (DSG) for 45 minutes and then in 1% paraformaldehyde

for 15 minutes at room temperature. Fixed cells were lysed in 10 ml of Lysis Buffer
1 [50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA, 0.1% IGEPAL 630 (Sigma
Aldrich)], containing 0.05% Triton X100, 2.5 % glycerol and supplemented with 1X
protease inhibitor cocktail (Roche) for 10 minutes on ice, followed by incubation in Buffer
2 [0.1 M Tris HCl (pH 8) and 200 mM NaCl with protease inhibitors] for 15 minutes
at room temperature. Chromatin was sonicated at 30% of amplitude for 10 minutes (10
cycles of 1 minute). The samples were centrifuged (2X 14,000 g for 5 minutes each), and
soluble chromatin was transferred to a fresh tube. Crosslinked DNA after sonication was
precipitated with 5μg of anti-YAP1 antibody (sc-15407X, Santa Cruz Biotechnology) or
non-immune rabbit IgG (ab27472, Abcam) overnight at 4°C. Chromatin/antibody complex
was pulled down with PureProteome™ Protein G Magnetic Beads (Millipore) and washed
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in the low- and high-salt buffers. After crosslinking reversion (65°C for 4 hours) and
Proteinase K treatment, chromatin was purified by phenol-chloroform extraction and
ethanol precipitation. Precipitated DNA was analyzed by qPCR using primers generated
for predicted TEAD binding sites or a non-relevant genomic region (Control).

ChIP-Seq Analysis—Human gene coordinates from genome assembly GRCh38 were

obtained from Ensembl BioMart25. Publicly available conservative IDR-thresholded ChIP-
Seq peak calls for TEAD1, TEAD3, and TEAD4 in cells were downloaded from the
ENCODE Project26. Genes were considered to have a TEAD binding site if at least
one ChIP-Seq peak overlapped with the region beginning 1kb upstream of the gene start
and ending 100 (m1000-p100) bases downstream of the gene start. Plots were created in
R v4.0.2 using the ggplot package; y axes show fold change of TEAD factor binding
versus control from ChIP-Seq data, also obtained from ENCODE; x axes show genomic
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coordinates. The promoter region is shown in gray and the gene region is shown in black.
Calculated peaks are shown in red. Hypergeometric enrichment p-values were calculated in
Python 3.8 using the scipy package.

KEGG Analysis—KEGG gene sets were downloaded from the Broad Institute’s
Molecular Signatures Database (MSigDB). Hypergeometric p-values for enrichment of gene
sets for predicted TEAD targets (as defined above) were calculated in Python 3.8 using the
scipy package.

Matrix remodeling experiments—Following treatment, PAAFs were plated in normal

growth medium supplemented with 0.5% FBS and 50 μg/ml of ascorbic acid. Medium was
changed every day. After five days, PAAF were removed by adding 1 ml of pre-warmed
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(37°C) extraction buffer (PBS/0.5% Triton X100/20 mM NH4OH). Matrices were washed
3 times (PBS). Naïve PAECs or PASMCs were then plated on top of these matrices
supplemented with normal growth medium 0.5% FBS or harvested for assessment of
collagen (Sircol assay) or fibrillar collagen content (Picrosirius Red stain). Twenty-four
hours later, cells were harvested for immunofluorescence experiments.

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 Rachedi et al. Page 20

Immunofluorescence—After the different treatment cells were fixed with PBS/PFA 4%

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for 10 min and permeabilized with PBS/Triton 100X 0.1% for 10min. Then cells were
incubated with anti-P-MLC2 (#3671; 1/100; Cell signaling) or anti-Ki67 (ab15580; 1/300;
Abcam) at room temperature for 2 hours. Secondary antibodies coupled with Alexa-594
(Thermo Scientific) or Alexa-488 were used at 1:500. Nuclei were counterstained with
DAPI (Sigma-Aldrich). Pictures were obtained using a ZEISS LSM Exciter confocal
microscope.

Measurement of collagen content in matrix—PAAF-derived matrix was harvested

and collagen cconcentration of the ECM was assessed using Sircol Soluble Collagen Assay
Kit (Biocolor) with a colorimetric reaction (measured at 550 nm) and a collagen reference
standard curve.

Measurement of collagen content in lung tissue—This protocol was adapted from

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a previously published protocol3. Rodent lung was weighed, minced, and incubated in
0.5 M acetic acid at 4°C. After overnight digestion, the acetic acid-soluble and insoluble
fractions were isolated by centrifugation. The soluble fraction was stored at −80°C, while
the insoluble fraction was digested by overnight incubation in 6M hydrochloric acid at 85°C.
Concentrations of soluble and insoluble (gelatinous) collagen fractions were determined
using a Sircol Soluble Collagen Assay Kit (Biocolor) with a colorimetric reaction (measured
at 550 nm) and a collagen reference standard curve.

Picrosirius red stain and quantification—Picrosirius red stain was achieved through
the use of 5μm paraffin sections stained with 0.1% picrosirius red (Direct Red80, Sigma-
Aldrich) and counterstained with Weigert’s hematoxylin to reveal fibrillar collagen. The
sections were then serially imaged using with an analyzer and polarizer oriented parallel
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and orthogonal to each other. Microscope conditions (lamp brightness, condenser opening,
objective, zoom, exposure time, and gain parameters) were constant throughout the imaging
of all samples. A minimal threshold was set on appropriate control sections for each
experiment in which only the light passing through the orthogonally-oriented polarizers
representing fibrous structures (i.e., excluding residual light from the black background) was
included. The threshold was maintained for all images across all conditions within each
experiment. The area of the transferred regions that was covered by the thresholded light
was calculated and at least 10 sections/vessel per condition were averaged together (Image J
software).

Atomic force microscopy—Rodent lungs were inflated with 0.025g of OCT by g of

body weight, frozen on liquid nitrogen vapor and store at −80°C. Rodent lung slices (10
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μm thickness) were cut out from their glass slide and the fragment of glass containing the
sample was glued on the bottom of a 50 mm dish (Willco Glass Bottom Dish). Before
measurements the sample was first rinsed and after covered with 4 ml of PBS 1x. The
mechanical properties of the samples were studied using a BioScope Catalyst atomic force
microscope (Bruker) coupled with and optical microscope (Leica DMI6000B) that enables,
by phase contrast, to pinpoint the areas of interest. For each sample, 8–12 vessels per
rodent (< 100 μm diameter) were analyzed using the “Point and Shoot” method, collecting

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 Rachedi et al. Page 21

from 80 to 120 force-distance curves at just as many discrete points. The experiments of
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microindentation were performed in PBS using a probe with a Borosilicate Glass spherical
tip (5 μm of diameter) and a cantilever with a nominal spring constant of 0.06 N/m
(Novascan). Indentations were carried out using a velocity of 2 μm/s, in relative trigger
mode and by setting the trigger threshold to 2 nN. The apparent Young’s (elastic) modulus
was calculated using the NanoScope Analysis 1.50 software (Bruker), fitting the force
curves to the Hertz spherical indentation model and using a Poisson’s ratio of 0.4. To
avoid large indentation, a minimum and a maximum Force Fit Boundary of 5% and 25%
respectively of the whole force curve was taken into account for the fit.

Statistical analysis—Numerical quantifications of experiments using cultured cells

represents mean ± standard deviation (SD), while numerical quantifications of experiments
using rodent or human reagents represent mean ± standard error of the mean (SEM).
Atomic force microscopy data are represented by Tukey box-and-whisker plots. Normality
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of data distribution was determined by Shapiro Wilk testing. Means of 2 sample groups
were compared by paired (when appropriate) and unpaired two-tailed Student’s T-test
for normally distributed data, while Mann-Whitney U non-parametric testing was used
for non-normally distributed data. For comparisons among different groups, analysis of
variance tests (ANOVA) followed by Bonferroni’s post-hoc analysis or Kruskal Wallis tests
with Dunn’s post-hoc analysis (where appropriate for non-normally distributed data) were
performed. A P-value less than 0.05 was considered significant.

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Author Manuscript

Acknowledgements:
We thank the B. Mari team members for advice and discussions as well as F. Aguila for artwork. The authors
acknowledge the “Microscopie Imagerie Côte d’Azur” (MICA), GIS-IBISA multi-sites platform and particularly
the IPMC, C3M and IRCAN (Molecular and Cellular Imaging facility PICMI) partners. This platform is supported
by the GIS IBiSA, Conseil Départemental 06, Région PACA ARC, Cancerôpole PACA,)”. We thank the Organic
Synthesis Core (Dr George Sukenick and Ms. Rong Wang) and the NMR Analytical Core (MSKCC) for help with
NMR and LC-MS experiments.

Funding:
This work was supported by: the French National Research Agency ANR-18-CE14-0025, ANR-21-CE44-0036 (for
T.B.) and ANR-20-CE14-0006 (for T.B. and F.P.); NIH grants R01 HL124021, HL122596, HL151228 as well as
the AHA grant 18EIA33900027 (S.Y.C.). The Organic Synthesis Core at MSKCC was supported in part by NCI
R50 CA243895-03 and NCI P30 CA008748-55.
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Highlights
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• YAP/TAZ control glutamine and serine catabolism for collagen biosynthesis

• Such amino acid metabolism promotes pathogenic activation of vascular

fibroblasts

• Pharmacologic or dietary intervention improves pulmonary vascular stiffness

and disease
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 Rachedi et al. Page 27
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Figure 1: In vitro and in vivo evidence of proline and glycine metabolic rewiring in PH.
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(A-B) Heatmap (A) and pathway enrichment analysis (B) of significantly (FDR<0.1;
P<0.05) modulated intracellular metabolites in PH-PAAFs, with log2(fold change) range
between −4 to 4. (C) Heatmap of significantly (FDR<0.1; P<0.05) modulated metabolites
in PH-PAAF conditioned media, with log2(fold change) range between −2 to 2. (D)
Schema of blood sampling for measurement of transpulmonary gradient of metabolites
in rat (yellow, site of blood collection). (E) Heat map of significantly (FDR<0.1; P<0.05)
modulated metabolites in the transpulmonary gradient of monocrotaline-exposed rats (N=8

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 Rachedi et al. Page 28

per group), with log2(fold change) range between −1 to 1. (F) Schema of blood sampling for
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measurement of transpulmonary gradient of metabolites in human. (G) Level of indicated

metabolites in the transpulmonary gradient of patients with PH (N=8) and control (N=5).
P values calculated by paired two-tailed Student’s T-test. Mono: Monocrotaline. PCW:
Pulmonary capillary wedge. N’ numbers indicate biological replicates.
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Figure 2: Glutamine and serine catabolism sustain proline and glycine anabolism in activated
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pulmonary arterial adventitial fibroblasts (PAAFs).

(A) Schematic representation of glucose, glutamine, and serine pathways. (B-E) PAAFs
plated on the indicated substrate and treated as indicated. Heatmap of 13C6-glucose (B),
13C -glutamine (C), 13C -glutamate (D) and 13C -serine (E) incorporation in intracellular
5 5 3
metabolites. (F) 18F-FGln-PET imaging displayed increased tracer uptake in RV (white
arrow) and lung (red arrow) of MCT PAH rats. (G-H) 18F-FGln uptake in regions of
bronchovasculature (G), and lung parenchyma (H) in monocrotaline-exposed rats (N=6)

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 Rachedi et al. Page 30

as compared with PBS control (N=6). Data are expressed as standard uptake value ratios
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(SUVr). (I-M) After treating monocrotaline-exposed rats vs. controls (N=3–4 per group)
with 15N-labeled glutamine, 15N-labeled serine, or 15N-labeled serine and 2H-labeled
glutamine administered simultaneously, rats were sacrificed, and fixed lung was analyzed
by multi-isotope imaging mass spectrometry (MIMS). Representative images (I, K and L)
and quantification of 15N-glutamine (J), and 15N-serine and 2H-glutamine (M) incorporation
by pulmonary arteriolar mural cells, expressed as percent above background ratio. The
rainbow scale is set from 0% above background ratio in blue to 100% for glutamine or
150% for serine in red (2 to 2.5 fold above background ratio). Scale=10 μm. In all the
panels *P<0.05; **P<0.01; two-tailed Student’s t-test was used; data show mean ± s.e.m. N’
numbers indicate biological replicates.
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Figure 3: Glutamine and serine catabolism sustain the metabolic needs of collagen biosynthesis.
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(A-B) Collagen concentration (media) of IL-6-activated PAAFs (A) and PH-PAAFs (B)
treated as indicated. (C-I) Schema of the experimental procedure (C). Representative image
(D and F) and quantification (E, G and I) of fibrillar collagen and vascular (PAEC and
PASMC) cell proliferation. (H) Matrix stiffness assessed by atomic force microscopy. Data
are represented by Tukey box-and-whisker plots. Median represents measures from n=3
matrices/group. In all panels **P<0.01; ***P<0.001; ANOVA with Bonferroni’s multiple
comparison test was used in (A-G, I); Kruskal-Wallis with Dunn’s post-hoc testing was

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used in (H); data show mean ± s.d. Scale=20 μm. For all the panels, each dot represents a
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biological replicates.
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Figure 4: YAP and TAZ metabolically control collagen biosynthesis.

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(A) Pathway enrichment analysis of genes with TEAD biding sites (ENCODE Project)
in their promoter regions. (B) Schematic of the collagen metabolism pathway. (C) TEAD
binding enrichment in the promoter region of indicated genes. (D) ChIP-qPCR confirmed
the presence of TEAD/YAP binding sites in the promoter regions of indicated genes.
Results are expressed as percentage of total input DNA prior to immunoprecipitation with
anti-YAP or anti-IgG control. (E-F) Heatmap with log2(fold change) range between −2 to 2
(E) and pathway enrichment analysis (F) of significantly (FDR<0.1; P<0.05) modulated

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 Rachedi et al. Page 34

intracellular metabolites in PAAFs treated as indicated. (G-H) PAAFs were treated as

indicated. Heatmap of 13C5-glutamine (G) and 13C3-serine (H) incorporation in intracellular
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metabolites. (I) Matrix collagen concentration of PAAFs treated as indicated. (J-L) Schema
of the experimental procedure (J). Representative image (K) and quantification (L) of
fibrillar collagen and vascular (PAEC and PASMC) cell proliferation. Scale=20 μm. In all
panels *P<0.05; **P<0.01; ****P<0.0001; Mann-Whitney test was used in (D:SLC6A9-E;
SHMT1:E); two-tailed Student’s T-test was used in remaining panels of (D); ANOVA with
Bonferroni’s multiple comparison test was used in (I, L: Ki67 graphs); Kruskal-Wallis with
Dunn’s post-hoc testing was used in (L: area thresholded). Data show mean ± s.d. For all the
panels, each dot represents a biological replicates.
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Figure 5: Genetic deletion of YAP and TAZ in vascular fibroblast prevents collagen biosynthesis
and ameliorates PH.
(A) Protocol for conditional YAP/TAZ deletion (cKO) in IL-6 transgenic mice. (B-C, E,
and H) Immunostaining and quantification for GLS1 (B), SHMT1 (C), collagen I (Col1;
E) and Ki67 (H) in pulmonary artery from YAP/TAZcKO mice or control mice (Ctrl).
(D) Collagen concentration (Total and fibrillar) of lungs from YAP/TAZcKO mice or Ctrl
mice. (F) Representative images and quantification of picrosirius red staining in pulmonary
artery from YAP/TAZcKO mice or Ctrl mice. (G) Pulmonary vascular stiffness assessed by
atomic force microscopy. Data are represented by Tukey box-and-whisker plots. Median
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represents measures from n=3 mice/group. (I-K) Pulmonary arteriolar muscularization (I),
right ventricular systolic pressure (J), and right ventricular hypertrophy (Fulton index,
RV/LV+S; K) of YAP/TAZcKO mice or Ctrl mice. Scale=50 μm. In all panels, *P<0.05;
**P<0.01; ***P<0.001; ****P<0.0001; Mann-Whitney test was used in (F:Orthogonal;
G-I); two-tailed Student’s T-test was used in (B-E; F:Parallel; J-K). Data show mean ± s.e.m.
Panel B-C, E-F, and H-I: each dot represents a single artery from N=5 Control mice and N=6
cKO mice. Panel D and J-K each dot represents a single mouse

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Figure 6: Pharmacologic inhibition of glutamine and serine catabolism decreases collagen

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biosynthesis and vascular stiffening in PH.

(A) In a protocol of disease reversion, rats were subjected to monocrotaline injection,
and seven days later, they received daily i.p. injections of either GLS1 inhibitor (CB-839,
N=8), or SHMT1 inhibitor (SHIN1, N=9), or both CB-839 + SHIN1 (N=9) or vehicle
(Ctrl; N=9). (B) Collagen concentration (Total and fibrillar) of lungs from rats treated
as indicated. (C) Immunostaining and quantification for collagen I (Col1) in pulmonary
artery from rats treated as indicated. (D) Pulmonary vascular stiffness assessed by atomic

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 Rachedi et al. Page 37

force microscopy. Data are represented by Tukey box-and-whisker plots. Median represents
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measures from n=4 rats/group for (SHIN1 and CB-839+SHIN1) and n=7 rats/group for (Ctrl
and CB-839). (E) Immunostaining and quantification for Ki67 in pulmonary artery from
rats treated as indicated. (F-H) Pulmonary arteriolar muscularization (F), right ventricular
systolic pressure (G), and right ventricular hypertrophy (Fulton index, RV/LV+S; H) of rats
treated as indicated. Each dot represents a single rat. Scale=50 μm. In all panels *P<0.05;
**P<0.01; ***P<0.001; ****P<0.0001; ANOVA with Bonferroni’s multiple comparison test
was used in (B, C, E, H); Kruskal-Wallis with Dunn’s post-hoc testing was used in (D, F, G).
Data show mean ± s.e.m. In panels B-C, and E-H each dot represents a single rat
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Figure 7: A glutamine- and serine-free diet prevents pulmonary vascular stiffening and PH.
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(A) Rats received normal chow until 12 weeks of age; they were then transferred to either
a control diet (synthetic diet, containing glutamine and serine) or a matched diet lacking
glutamine and serine (Gln−/Ser− diet). Eighteen (18) days after diets were initiated, rats
were injected with monocrotaline. At Day 39, rats were euthanized and assessed for PH. (B)
Serum levels of glutamine, serine, and glucose were analyzed at serial time points. Mean
expression in control group was assigned a fold change of 1, to which relevant samples were
compared. (C) At D39, serum levels of the indicated metabolites were analyzed. Unpaired

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 Rachedi et al. Page 39

two-tailed Student’s T-test and Mann-Whitney test. (D) Collagen concentration (total and
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fibrillar) of lungs from rats treated as indicated. (E) Immunostaining and quantification
for collagen I (Col1) in pulmonary artery from rats treated as indicated. (F) Pulmonary
vascular stiffness assessed by atomic force microscopy. Data are represented by Tukey
box-and-whisker plots. Median represents measures from n=7 rats/group for (standard diet)
and n=4 rats/group for (synthetic diet and Gln−/Ser− diet). (G-H) Immunostaining (G)
and quantification (H) for Ki67 in pulmonary artery from rats treated as indicated. (I-K)
Pulmonary arteriolar muscularization (I), right ventricular systolic pressure (J), and right
ventricular hypertrophy (Fulton index, RV/LV+S; K) of rats treated as indicated. Each
dot represents a single rat. Scale=50 μm. In all panels, *P<0.05; **P<0.01; ***P<0.001;
****P<0.0001; Mann-Whitney test was used in (C:glutamine/glutamate; phenylalanine/
tyrosine; D:soluble/insoluble collagen); two-tailed Student’s T-test was used in remaining
panels of (C) as well as (D: collagen). ANOVA with Bonferroni’s multiple comparison test
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was used in (E, G, H, I, J); Kruskal-Wallis with Dunn’s post-hoc testing was used in (F).
Data show mean ± s.e.m. In panels C-E and G-J each dot represents a single rat.
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KEY RESOURCES TABLE

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REAGENT or RESOURCE SOURCE IDENTIFIER

Antibodies
Rabbit Polyclonal anti-MLC2 Cell Signaling Technology Cat# 3672S; RRID: AB_10692513

Mouse Monoclonal anti-P-MLC2 Cell Signaling Technology Cat# 3675S; RRID: AB_2250969

Mouse Monoclonal anti-HIF1-α Abcam Cat# ab1; RRID: AB_296474

Mouse Monoclonal anti-Tubulin Santa Cruz Biotechnology Cat#sc-398937

Mouse Monoclonal anti-Hsp90 Santa Cruz Biotechnology Cat# sc-69703; RRID: AB_2121191

Rabbit Polyclonal anti-GLS1 Abcam Cat# ab156876; RRID: AB_2721038

Rabbit Monoclonal anti-SHMT1 Cell Signaling Technology Cat# 80715; RRID: AB_2799957

Rabbit Monoclonal anti-YAP Cell Signaling Technology Cat# 14074; RRID: AB_2650491

Rabbit Polyclonal anti-YAP Santa Cruz Biotechnology Cat# sc-15407; RRID: AB_2273277

Rabbit Polyclonal anti-TAZ Santa Cruz Biotechnology Cat# sc-48805; RRID: AB_2216639
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Rabbit Polyclonal anti-Ki67 Abcam Cat# ab15580; RRID: AB_443209

Goat Polyclonal anti-αSMA Abcam Cat# ab21027; RRID: AB_1951138

Mouse Monoclonal anti-αSMA Sigma-Aldrich Cat# A5228–200; RRID: AB_262054

Rabbit Monoclonal anti- αSMA Abcam Cat# ab32575; RRID: AB_722538

Mouse Monoclonal anti-Vimentin Abcam Cat# ab20346; RRID: AB_445527

Goat Monoclonal anti-CD31 R&D Systems Cat# AF3628; RRID: AB_2161028

Rabbit Monoclonal anti-Collagen I Abcam Cat#ab260043

Peroxidase-AffiniPure Donkey Anti-Mouse IgG (H+L) Jackson ImmunoResearch Labs Cat# 715–035-150; RRID:
AB_2340770

Peroxidase-AffiniPure Donkey Anti-Rabbit IgG (H+L) Jackson ImmunoResearch Labs Cat# 711–035-152; RRID:
AB_10015282

Donkey anti-Goat IgG (H+L) Antibody, Alexa Fluor 488 Thermo Fisher Scientific Cat# A11055; RRID: AB_2534102
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Donkey anti-Rabbit IgG (H+L) Antibody, Alexa Fluor 594 Thermo Fisher Scientific Cat# A21207; RRID: AB_141637

Donkey anti-Mouse IgG (H+L) Antibody, Alexa Fluor 647 Thermo Fisher Scientific Cat# A31571; RRID: AB_162542

Bacterial and virus strains

/ / /

Biological samples

Chemicals, peptides, and recombinant proteins

Monocrotaline Sigma-Aldrich Cat#PHL89251

Tamoxifen Sigma-Aldrich Cat#T5648

Sirius red (Direct Red 80) Sigma-Aldrich Cat#365548

DAPI Sigma-Aldrich Cat#D9542

Recombinant Human TGF-β1 PeproTech Cat#100–21

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Recombinant Human IL-6 PeproTech Cat#200–06

CB-839 glutaminase inhibitor Selleckchem Cat#S7655

SHIN 1 SHMT inhibitor Tocris Cat#6998

D-Glucose U-13C6 Cambridge Isotope Laboratories Cat#CLM-1396–1

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REAGENT or RESOURCE SOURCE IDENTIFIER

L-Glutamine 13C Cambridge Isotope Laboratories Cat#CLM-1822-H-0.1

5
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L-Glutamate 13C5 Cambridge Isotope Laboratories Cat#CLM-1800-H-0.5

L-Serine 13C3 Cambridge Isotope Laboratories Cat#CLM-1574-H-0.1

L-Proline 13C5 Cambridge Isotope Laboratories Cat#CLM-2260-H-0.1

Lipofectamine 2000 Thermo Fisher Scientific Cat#11668500

Pierce RIPA Buffer Thermo Fisher Scientific Cat#89900

2x Laemmli Sample Buffer Bio-Rad Cat#1610737

Immobilon Western (HRP Substrate) Merck Cat#WBKLS0500

QIAzol Lysis Reagent Qiagen Cat#79306

Critical commercial assays

Sircol Soluble Collagen assay kit Biocolor Cat#S1000
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miRNeasy Kit Qiagen Cat#217084

MultiScribe Reverse Transcriptase Kit Thermo Fisher Scientific Cat#4311235

Deposited data

Experimental models: Cell lines

Human Pulmonary Artery Adventitial Fibroblasts (hPAAF) Sciencell Cat#3120

Human Pulmonary Artery Smooth Muscle Cells (hPASMC) Lonza Cat#CC-2581

Human Pulmonary Artery Endothelial Cells (hPAEC) Lonza Cat#CC-2530

hPAAF from Pulmonary Hypertension donors (PH-PAAF) French Network on Pulmonary /

Hypertension

HEK293T ATCC CRL-3216

Experimental models: Organisms/strains

Mouse: Col1a2-Cre-ER The Jackson Laboratory Strain # 029235
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Mouse: Yap1 fl/fl/Tazfl/fl The Jackson Laboratory Strain #:030532

Mouse: CC10-IL-6 Tg(+) × C57/BL6 Tg(−) Gift from Dr Aaron B. Waxman

Rat: Male Sprague-Dawley rat Charles River Laboratories

Oligonucleotides
siRNA Negative control Horizon Discovery Cat#D-001810–10

siRNA GLS1_pool Horizon Discovery Cat#L-004548–01

siRNA GLS1_1: CCUGAAGCAGUUCGAAAUA Horizon Discovery Cat#J-004548–09

siRNA GLS1_2: CUGAAUAUGUGCAUCGAUA Horizon Discovery Cat#J-004548–10

siRNA SHMT1_pool Horizon Discovery Cat#L-004617–00

siRNA SHMT1_1: GAGCUGGCAUGAUCUUCUA Horizon Discovery Cat#J-004617–05

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siRNA SHMT1_2: CCUAGGCUCUUGCUUAAAU Horizon Discovery Cat#J-004617–06

siRNA YAP1_pool Horizon Discovery Cat#L-012200–00

siRNA YAP1_1: GGUCAGAGAUACUUCUUA Horizon Discovery Cat#J-012200–07

siRNA YAP1_2: GAACAUAGAAGGAGAGGAG Horizon Discovery Cat#J-012200–08

siRNA TAZ_pool Horizon Discovery Cat#L-016083–00

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REAGENT or RESOURCE SOURCE IDENTIFIER

siRNA TAZ_1 : GACAUGAGAUCCAUCACUA Horizon Discovery Cat#J-016083–05
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siRNA TAZ_2: GGACAAACACCCAUGAACA Horizon Discovery Cat#J-016083–06

Recombinant DNA
Human: pcDNA Flag Yap1 Levy et al., 2008 Addgene; Plasmid #18881

Vector: pCDH-CMV-MCS-EF1-copGFP System Biosciences Cat#CD511B-1

Software and algorithms

GraphPad Prism 6 GraphPad software https://www.graphpad.com/scientific-
software/prism/

ImageJ NIH https://imagej-nih-gov.gate2.inist.fr/ij/

TraceFinder 2.1 Thermo Fisher Scientific /

Xcalibur 4.0 Thermo Fisher Scientific /

NanoScope Analysis 1.50 Bruker /

Other (Diets)
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Synthetic diet-control SAFE /

Glutamine and Serine-free diet (Glycine (-) /Proline (-) / SAFE /

Glutamine (-) /Serine (-))

Deposited Data
Data S1 This paper
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