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Circulation. 2021;143:2091–2109. DOI: 10.1161/CIRCULATIONAHA.120.051171 May 25, 2021 2091
Circulation
Jorge Oller , PhD
Enrique
Gabandé-Rodríguez ,
PhD*
María Jesús Ruiz-Rodríguez,
BS*
Gabriela Desdín-Micó , BS
Juan Francisco Aranda, PhD
Raquel Rodrigues-Diez , PhD
Constanza
Ballesteros-Martínez ,
PhD
Eva María Blanco
Raquel Roldan-Montero, BS
Pedro Acuña, BS
Alberto Forteza Gil, MD, PhD
Carlos E. Martín-López, MD,
PhD
J. Francisco Nistal, MD, PhD
Christian L. Lino Cardenas,
PharmD, MSc, PhD
Mark Evan Lindsay, MD, PhD
José Luís Martín-Ventura, PhD
Ana M. Briones, PhD
Juan Miguel Redondo , PhD
María Mittelbrunn , PhD
https://www.ahajournals.org/journal/circ
*E. Gabandé-Rodríguez and M.J.
Ruiz-Rodriguez contributed equally.
Key Words: aortic aneurysm ◼ DNA,
mitochondrial ◼ extracellular matrix
◼ genetic diseases, inborn ◼ glycolysis
◼ Marfan syndrome ◼ muscle, smooth,
vascular
Sources of Funding, see page 2108
© 2021 The Authors. Circulation is
published on behalf of the American
Heart Association, Inc., by Wolters
Kluwer Health, Inc. This is an open
access article under the terms of the
Creative Commons Attribution License,
which permits use, distribution, and
reproduction in any medium, provided
that the original work is properly cited.
BACKGROUND: Marfan syndrome (MFS) is an autosomal dominant
disorder of the connective tissue caused by mutations in the FBN1
(fibrillin-1) gene encoding a large glycoprotein in the extracellular matrix
called fibrillin-1. The major complication of this connective disorder
is the risk to develop thoracic aortic aneurysm. To date, no effective
pharmacologic therapies have been identified for the management
of thoracic aortic disease and the only options capable of preventing
aneurysm rupture are endovascular repair or open surgery. Here, we have
studied the role of mitochondrial dysfunction in the progression of thoracic
aortic aneurysm and mitochondrial boosting strategies as a potential
treatment to managing aortic aneurysms.
METHODS: Combining transcriptomics and metabolic analysis of aortas
from an MFS mouse model (Fbn1c1039g/+) and MFS patients, we have identified
mitochondrial dysfunction alongside with mtDNA depletion as a new
hallmark of aortic aneurysm disease in MFS. To demonstrate the importance
of mitochondrial decline in the development of aneurysms, we generated
a conditional mouse model with mitochondrial dysfunction specifically
in vascular smooth muscle cells (VSMC) by conditional depleting Tfam
(mitochondrial transcription factor A; Myh11-CreERT2Tfamflox/flox mice). We used
a mouse model of MFS to test for drugs that can revert aortic disease by
enhancing Tfam levels and mitochondrial respiration.
RESULTS: The main canonical pathways highlighted in the transcriptomic
analysis in aortas from Fbn1c1039g/+ mice were those related to metabolic
function, such as mitochondrial dysfunction. Mitochondrial complexes,
whose transcription depends on Tfam and mitochondrial DNA content,
were reduced in aortas from young Fbn1c1039g/+ mice. In vitro experiments
in Fbn1-silenced VSMCs presented increased lactate production and
decreased oxygen consumption. Similar results were found in MFS patients.
VSMCs seeded in matrices produced by Fbn1-deficient VSMCs undergo
mitochondrial dysfunction. Conditional Tfam-deficient VSMC mice lose
their contractile capacity, showed aortic aneurysms, and died prematurely.
Restoring mitochondrial metabolism with the NAD precursor nicotinamide
riboside rapidly reverses aortic aneurysm in Fbn1c1039g/+ mice.
CONCLUSIONS: Mitochondrial function of VSMCs is controlled by the
extracellular matrix and drives the development of aortic aneurysm in Marfan
syndrome. Targeting vascular metabolism is a new available therapeutic
strategy for managing aortic aneurysms associated with genetic disorders.
Extracellular Tuning of Mitochondrial
Respiration Leads to Aortic Aneurysm
ORIGINAL RESEARCH ARTICLE
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
May 25, 2021 Circulation. 2021;143:2091–2109. DOI: 10.1161/CIRCULATIONAHA.120.051171
2092
ORIGINAL RESEARCH
ARTICLE
Different inherited disorders affect the structure of
the arterial wall and lead to thoracic aortic aneu-
rysms (TAA).1,2 Most of these diseases are associ-
ated with mutations in genes related to the extracel-
lular matrix (ECM), or the vascular smooth muscle cell
(VSMC)–contractile apparatus. Marfan syndrome (MFS)
is one of the most commonly inherited disorders affect-
ing connective tissue. It is caused by mutations in the
gene that encodes for the extracellular protein FBN1
(fibrillin-1) and has a reported incidence of 1 in 5000
individuals.3,4 MFS patients present extended bones,
lens luxation, and decreased life expectancy primarily
attributable to TAAs.5 Mutations in genes that encode
proteins involved in TGFβ–signaling cause Loeys–Dietz
syndrome, a disease that phenocopies part of the skel-
etal features of MFS.6 Vascular-type Ehlers–Danlos syn-
drome is mainly caused by mutations in COL3A1 gene
(collagen 3a1) that produce hyperelasticity in joints and
skin, small stature, and aortic dissections.7 Moreover,
the rare condition Cutis laxa syndrome is caused by
mutations in elastin fibers genes (ELN [elastin], FBLN4
[fibulin 4], FBLN5 [fibulin 5]) that produce a loss of elas-
tic properties in tissues such as the skin and arteries.8,9
Finally, although most of the genes involved in familial
forms of nonsyndromic thoracic aortic aneurysm and
dissections remain unknown, some of those identified
(accounting for only about 20% of all patients with fa-
milial forms of nonsyndromic thoracic aortic aneurysm
and dissections ) are involved in the maintenance of the
smooth muscle contractile apparatus, such as PRKG1
(cGMP-dependent protein kinase 1), MYLK (myosin
light chain kinase), MYH11 (myosin heavy chain11),
and ACTA2 (actin α2, smooth muscle).10
The major complication of all these connective inher-
ited disorders is the risk to develop TAAs. TAA is a com-
plex vascular pathology characterized by permanent
dilation of the thoracic aorta. TAA progression can lead
to catastrophic consequences like rupture or dissection
of the arterial wall, causing patient death because of
extensive hemorrhage. VSMCs are located in the me-
dial layer of arteries and directly drive the contraction
of the vascular wall regulating the size of the vessel lu-
men. VSMCs can shift reversibly from a quiescent or
contractile phenotype to a secretory phenotype.11–13
Changes toward this secretory phenotype generate
pathologic features such as an elevated proliferation
rate and increased ECM accumulation favoring medial
degeneration in the aneurysm development.12–15 ECM
remodeling in the aortic wall results in increased aortic
stiffness.16–18 Studies have started to reveal the connec-
tion between cellular adhesion and cytoskeletal reorga-
nization and the metabolism of the cell,19–21 supporting
that the composition and thestiffness of the ECM regu-
late cellular metabolism.22
Here, we investigate the implication of VSMC me-
tabolism in the progression and development of aortic
aneurysm in MFS. We provide evidence that mitochon-
drial metabolism is a key regulator of VSMC phenotype
during aortic remodeling and is fine-tuned by ECM
composition. To demonstrate the importance of mi-
tochondrial metabolism in the development of aneu-
rysms, we generated a conditional mouse model with
mitochondrial dysfunction specifically in VSMCs by de-
pleting Tfam (mitochondrial transcription factor A). As
Tfam controls the transcription, replication, and stabil-
ity of mitochondrial DNA (mtDNA),23 depleting Tfam
has been proved to be an effective approach to induce
severe mitochondrial dysfunction in different cells and
tissues.24–27 Tfam-deficient VSMC mice developed aor-
tic dilation, medial degeneration, aortic aneurysm, and
fatal dissections, further supporting that mitochondrial
function is a key regulator of the VSMC phenotype dur-
ing aortic remodeling. Finally, we assessed the thera-
peutic potential of boosting mitochondrial metabolism
in MFS. Our results indicate that mitochondrial func-
tion could be an effective target for pharmacologic ap-
proaches to managing aortic dilation and preventing
aortic dissection associated with genetic disorders.
METHODS
The data, analytic methods, and study materials that support
the findings of this study are available from the corresponding
author on reasonable request.
Mouse Strains and Animal Procedures
The Marfan mouse model, which harbors a Fbn1C1039G/+ allele
(JAX stock No. 012885), was previously described.28 For spe-
cific ablation of Tfam in smooth muscle, we crossed Tfamflox/
flox mice23 with mice carrying the Myh11-CreERT2 allele (JAX
Clinical Perspective
What Is New?
• Tfam and mitochondrial-DNA levels decline corre-
late with aortic dilation in Fbn1c1039g/+ mice.
• Aortic samples and cells from patients with Marfan
syndrome present mitochondrial defects.
• Mitochondrial respiration is fine-tuned by the
extracellular matrix.
• Mitochondrial dysfunction in vascular smooth mus-
cle cells promotes aortic dilation, aneurysms, and
premature death.
• Boosting mitochondrial function by a NAD+ precur-
sor rapidly reverses transcriptional signature and
aortic dilation in Fbn1c1039g/+ mice.
What Are the Clinical Implications?
• Boosting mitochondrial function with NAD+ pre-
cursors is a new therapeutic opportunity to man-
age aortic aneurysms associated with connective
genetic disorders and to prevent aortic dissection.
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
Circulation. 2021;143:2091–2109. DOI: 10.1161/CIRCULATIONAHA.120.051171 May 25, 2021 2093
ORIGINAL RESEARCH
ARTICLE
stock No. 019079) that expresses a tamoxifen-inducible Cre
recombinase under the regulatory sequences of smooth
muscle Myh11 promoter, inserted into Y-chromosome.23,29
Wild type littermates were used as controls for Marfan mice
and TfamWt/Wt Myh11-CreERT2 for Tfamflox/flox Myh11-CreERT2,
unless otherwise specified. For conditional gene deletion,
Myh11-CreERT2TfamWt/Wt and Myh11-CreERT2Tfamflox/flox 4- to
5-week-old male mice received daily 1-mg IP injections of
tamoxifen (Sigma-Aldrich) on 5 consecutive days. Ang II
(angiotensin II) dissolved in saline (Sigma-Aldrich) was infused
at 1 μg·kg−1·min−1 using subcutaneous osmotic minipumps
(Model 2004, Alzet Corp.). Nicotinamide riboside ([NR]
Novalix) was administered intraperitoneally at 1000 mg/kg in
saline every other day. Mice were housed at the pathogen-
free animal facility of the Centro Nacional de Investigaciones
Cardiovasculares Carlos III and Centro de Biología Molecular
Severo Ochoa following the animal care standards of the
institution. Animal procedures were approved by the Centro
Nacional de Investigaciones Cardiovasculares Carlos III and
Centro de Biología Molecular Severo Ochoa−Universidad
Autonoma de Madrid Ethics Committee and the Madrid
regional authorities (ref. PROEX 283/16) and conformed with
European Union Directive 2010/63EU and Recommendation
2007/526/EC regarding the protection of animals used
for experimental and other scientific purposes, enforced in
Spanish law under Real Decreto 1201/2005.
Blood Pressure Measurements and In
Vivo Imaging
Arterial blood pressure (BP) was measured by the mouse tail-
cuff method using the automated BP-2000 Blood Pressure
Analysis System (Visitech Systems, Apex, NC). Mice were
trained for BP measurements every day for 5 consecutive
days. After the training period, BP was measured 1 day before
treatment to determine the baseline BP values in each mouse
cohort. BP measurements were recorded in mice located in a
tail-cuff restrainer over a warmed surface (37°C). Fifteen con-
secutive systolic and diastolic BP measurements were made,
and the last 10 readings per mouse were recorded and aver-
aged. For in vivo ultrasound images, the aortic diameter was
monitored in isoflurane-anesthetized mice (2% isoflurane) by
high-frequency ultrasound with a VEVO 2100 echography
device (VisualSonics, Toronto, Canada) at 30-μ resolution.
Maximal internal aortic diameters were measured at diastole
using VEVO 2100 software, version 1.5.0.
Cell Procedures
Isolation and culture of primary mouse VSMCs were described
in Esteban et al.30 Tissue was digested with a solution of col-
lagenase and elastase (Worthington Biochem) until a single-
cell suspension was obtained. All experiments with primary
VSMCs were performed during passages 2 to 7. Lentiviral
transduction was performed in VSMCs during 5 hours at a
multiplicity of infection of 3. The medium was then replaced
with fresh DMEM supplemented with 10% FBS and cells were
cultured for 5 more days, treated with NR for 5 more days,
and then serum-starved for 16 hours. The HEK-293T (CRL-
1573) and Jurkat (Clone E6-1, TIB-152) cell lines, required
for high-titer lentivirus production and lentivirus titration,
respectively, were purchased from American Type Culture
Collection. Four apparently healthy male controls (GM00024,
GM01717, GM03652, GM23963) and 4 males with aged-
mismatched fibroblasts from MFS patients with aortic dissec-
tion, 2 with FBN1 point mutations (GM21499, GM21946),
and 2 with haploinsufficient FBN1 mutations (GM21983,
GM21978), were purchased to Coriell Cell Repositories. The
experiments were performed during passages 5 to 10. All
cells tested negative for Mycoplasma.
Lentivirus Production and Infection
The Cre and green fluorescent protein coding sequence was
obtained by polymerase chain reaction (PCR) amplification
and cloned into the pHRSIN lentiviral vector.31 Lentiviruses
expressing short hairpin RNA−targeting murine Fbn1, and
control short hairpin RNA were purchased from Sigma-
Aldrich. Pseudo-typed lentiviruses were produced by tran-
sient calcium phosphate transfection of HEK-293T cells and
concentrated from culture supernatant by ultracentrifuga-
tion (2 hours at 128 000g; Ultraclear Tubes; SW28 rotor and
Optima L-100 XP Ultracentrifuge; Beckman). Viruses were
suspended in cold sterile PBS and titrated by transduction
of Jurkat cells for 48 hours. Transduction efficiency (green
fluorescent protein−expressing cells and puromycin-resistant
cells) and cell death (propidium iodide staining) were quanti-
fied by flow cytometry.32
Extracellular Flux Analysis and
Extracellular L-Lactate Determination
Oxygen consumption rates (OCR) were measured in XF-96
Extracellular Flux Analyzers (Seahorse Bioscience) in 25 000
mouse aortic VSMCs or 50 000 human fibroblasts. Cells were
seeded in nonbuffered DMEM medium containing either
25 mM glucose or 1 mM CaCl2. Three measurements were
obtained under basal conditions and on addition of oligo-
mycin (1 mM), fluoro carbonyl cyanide phenylhydrazone
(1.5 mM), and rotenone (100 nmol/L) + antimycin A (1 mM).
OCR measurements were normalized to protein cell extracts.
Extracellular lactate determination was performed with sin-
gle-use reagent strips for Accutrend Plus Lactate Pro (Roche),
based on an enzymatic spectrophotometry system by lactate
oxidase layer. We analyzed 20 µL of conditioned medium
after 24 hours of culture. L-lactate was normalized to total
protein cell extracts.
β-Galactosidase Activity Assay
For the β-galactosidase quantitative assay, tissues were lysed
with T-PER Tissue Protein Extraction Reagent (78510; Thermo
Scientific). Lysates were centrifuged at 10 000g for 5 minutes
and the supernatant was collected. Fifty microliters of protein
lysates were mixed with 50 µL of Pierce β-galactosidase Assay
Reagent (75705; Thermo Scientific) for the assay. The reaction
was incubated for 1 hour and the absorbance was measured
at 415 nm. Values were normalized to total protein extracts.
ECM Assays
Experiments of ECM were based on Castelló-Cros et al.33 First,
culture plates were coated with 1% of gelatin (Sigma-Aldrich)
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
May 25, 2021 Circulation. 2021;143:2091–2109. DOI: 10.1161/CIRCULATIONAHA.120.051171
2094
ORIGINAL RESEARCH
ARTICLE
and 0.01% collagen I (Gibco) for 1 hour at 37ºC. The gelatin
was cross-linked by adding 1% glutaraldehyde in PBS and incu-
bated for 30 minutes at 37ºC. Then, the plates were washed
3 times for 5 minutes with PBS and 1 additional wash with
DMEM. Then, VSMCs were seeded at a confluence of 50% and
cultured for 5 days to allow production of the ECM. For the
decellularization step, ice-cold extraction buffer (0.2% of Triton
X-100, 20 mM NH4OH in DMEM) was added to the cells and
observed under a tissue culture microscope until the VSMCs
were completely lysed (5–10 minutes). Total cell lysis was tested
by analyzing 4′,6-diamidino-2-phenylindole staining in a cover-
slip. Then, control VSMCs were seeded in the ECMs at 100% of
confluence and the experiments were analyzed after 24 hours.
Real-Time and Quantitative PCR
Aortas were extracted after perfusion with 5 mL saline solution,
and the adventitia layer was discarded. Liquid nitrogen frozen
tissue was homogenized using a cold mortar and an automatic
bead homogenizer (MagNA Lyzer, Roche). Total RNA was iso-
lated with Trizol (Life Technologies). Total RNA (1 μg) was first
digested with DNAse and reverse-transcribed with Maxima First
Strand cDNA Synthesis Kit (ThermoFisher). For analysis of mtDNA
levels, total DNA from cells and tissues was extracted with the
SurePrep kit (Fisher Scientific) or Trizol, respectively, according to
the manufacture’s guidelines. DNA was amplified using primers
specific for cytochrome mt-Co1 (mitochondrially-encoded cyto-
chrome c oxidase 1) and mitochondrial 16S rRNA, then normal-
ized to B2M (β-2 macroglobulin) and H2K nuclear-encoded gene
controls. Real-time quantitative PCR (qPCR) was performed with
the primers indicated in Table I in the Data Supplement. qPCR
reactions were performed in triplicate with SYBR master mix
(Promega), according to the manufacturer’s guidelines. To exam-
ine probe specificity, we conducted a postamplification melting
curve analysis. For each reaction, only 1 melting temperature
peak was produced. The amount of target mRNA in samples
was estimated by the 2−CT relative quantification method, using
B2M, YWHAZ (tyrosine 3-monooxygenase/tryptophan 5-mono-
oxygenase activation protein ζ), and PP1A (protein phosphatase
1 catalytic α) for normalization. Fold ratios were calculated rela-
tive to mRNA expression levels from controls.
Library Preparation and Illumina
Sequencing
Aortas were extracted after perfusion of cold saline solution and
the adventitia layer was discarded. Frozen tissue was homog-
enized, and total RNA was isolated with Trizol (Roche). RNA
from Libraries were prepared according to the instructions of the
NEBNext Ultra Directional RNA Library prep kit for Illumina (New
England Biolabs), following the Poly(A) mRNA Magnetic Isolation
Module protocol. The input yield of total RNA to start the pro-
tocol was >300 ng quantified by an Agilent 2100 Bioanalyzer
using an RNA 6000 nano LabChip kit. The obtained libraries
were validated and quantified by an Agilent 2100 Bioanalyzer
using a DNA7500 LabChip kit and an equimolecular pool of
libraries was titrated by qPCR using the KAPA SYBR FAST qPCR
Kit for LightCycler 480 (Kapa BioSystems) and a reference stan-
dard for quantification. After processing on the Illumina HiSeq
2500 instrument, FastQ files were generated containing nucleo-
tide data and quality scores for each position. RNA-sequencing
reads were mapped to the Mus musculus reference genome,
GRCm38.p6, using Hisat2 v2.1.0 software. Reads were then
preprocessed with SAMtools v1.7 to transform Sequence
Alignment/Map files into Binary Alignment/Map files and sorted.
These files were used as an input for the HTSeq v0.6.1 pack-
age that produces a file containing the mapped reads per gene
(as defined by the Mus musculus, GRCm38.96 version, gff
file) for each sample. Once obtained HTSeq read counts, the
Bioconductor RNA-sequencing workflow was followed to detect
the expression differences of genes using the DESeq2 statistical
package. Ingenuity pathway analysis was used to identify cellular
biologic functions, gene clusters, and upstream regulators.
Immunoblot
For Western blot analysis, cells were lysed at 4ºC in radioim-
munoprecipitation assaybuffer containing protease and phos-
phatase inhibitors cocktail (Sigma). Proteins were separated
by SDS-PAGE and transferred onto 0.45-µm pore size polyvi-
nylidene fluoride membranes (Immobilon-P PVDF membrane;
Millipore). Polyvinylidene fluoride membranes were blocked
with TBS-T (50 mM Tris, 150 mM NaCl, and 0.1% Tween-
20) containing 5% (wt/vol) milk. Membranes were incubated
with primary antibodies diluted from 1/500 to 1/1000, fol-
lowed by TBS-T washes and incubation with HRP (horseradish
peroxidase)−conjugated secondary antibodies (GE Healthcare).
The signal was visualized by enhanced chemiluminescence
with Luminata Forte Western HRP Substrate (Millipore) and the
ImageQuant LAS 4000 imaging system. The following antibod-
ies were used: anti-TFAM (Proteintech), anti-MT-CO1 (Millipore),
anti-HIF1a and anti-mt-ND1 (Novus Biologicals), anti-P53 (Santa
Cruz), anti-PGC1a (Thermo Scientific), anti-VDAC (Abcam),
anti-Actin (Abcam), and anti–α-tubulin (Cell Signaling).
Aortic Histology
After euthanization by CO2 inhalation, mice were perfused with
saline. Aortas were then isolated and fixed in 10% formalin over-
night at 4°C. Paraffin cross-sections (5 μm) from fixed organs
were stained with Masson trichrome, Alcian blue, or Verhoeff
Van Gieson elastic, or they were used for immunohistochemistry
or immunofluorescence. Elastic fibers were stained with a modi-
fied Verhoeff Van Gieson elastin stain kit (Sigma-Aldrich). Elastic
lamina breaks, defined as interruptions in the elastic fibers, were
counted in the entire medial layer of 3 consecutive cross-sec-
tions per mouse, using 4 to 16 mice per experiment, and the
mean number of breaks was calculated. For immunostaining,
the deparaffinized sections were rehydrated, boiled to retrieve
antigens (10 mM citrate buffer, 0.05%Triton X-100, pH 6) and
blocked for 45 minutes with 10% goat normal serum, 5% horse
serum, 0.05% TritonX-100, and 2% BSA in PBS. Samples were
incubated with the following antibodies for immunohistochem-
istry or immunofluorescence: monoclonal anti-SMA (1/500,
C6198; Sigma), polyclonal anti-TFAM (1/300; Proteintech), poly-
clonal anti-MT-ND1 (1/300; Proteintech), monoclonal anti-MT-
CO1 (1/300; Invitrogen), polyclonal anti-HIF1A, and anti-MYC
(1/500; Novus Biologicals). Specificity was determined by sub-
stituting primary antibody with unrelated IgG (Santa Cruz). For
immunohistochemistry, endogenous peroxidase and biotin were
blocked with 1% hydrogen peroxide−methanol for 10 min-
utes and a biotin blocking kit (Vector Laboratories), respectively.
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
Circulation. 2021;143:2091–2109. DOI: 10.1161/CIRCULATIONAHA.120.051171 May 25, 2021 2095
ORIGINAL RESEARCH
ARTICLE
Color was developed in all samples at the same time with 3,
3-diaminobenzidine (DAB, Vector Laboratories), and sections
were counterstained with hematoxylin and mounted in DPX
(Fluka). For immunofluorescence, secondary antibodies were
Alexa Fluor 546−conjugated goat anti-rabbit and Alexa Fluor
647−conjugated goat anti-rabbit (BD Pharmingen). For F-actin
determination, fixed aortas were embedded in OCT (Tissue-Tek
Sakura), and 5-μm cross-sections were incubated for 30 minutes
with 1:1000 Phalloidin-657 (Millipore) after 10 minutes of incu-
bation with 0.3% Triton X-100 in PBS. Sections were mounted
with 4′,6-diamidino-2-phenylindole in Citifluor AF4 mounting
medium (Aname). Images were acquired at 1024×1024 pixels
and 8 bits using a Zeiss LSM800 microscope with 40× oil-immer-
sion objectives.
Vascular Reactivity
Aorta and mesenteric resistance arteries were dissected and
2-mm length segments were mounted in a wire myograph for
isometric tension recording. After a 30-minute equilibration
period in oxygenated Krebs−Henseleit solution at 37°C and
pH 7.4, segments were stretched to their optimal lumen diam-
eter for active tension development. Contractility of the seg-
ments was tested by an initial exposure to a high K+ solution
(120 mmol/L). After washing, a concentration−response curve
to increasing concentration of phenylephrine (1 nmol/L–50
µmol/L) and to U46619 (0.1 nmol/L–1 µmol/L) were carried out.
Gelatin Zymography
Supernatants form cell culture were prepared as described.32
Extracts (15 μg) were fractioned under nonreducing conditions
on 10% SDS–polyacrylamide gels containing 1% gelatin (Sigma).
Gels were washed 3 times in 2.5% Triton X-100 for 2 hours at
room temperature, incubated overnight at 37°C in 50 mM Tris-
HCl (pH 7.5), 10 mM CaCl2, and 200 mM NaCl, and stained with
Coomassie blue. The areas of gelatinolytic matrix metalloprotein-
ase (MMP) activity were visualized as transparent bands. Images
were analyzed with Quantity One software (Bio-Rad).
Human Samples
The study was approved by the Ethics and Clinical Research
Committee of Instituto de Salud Carlos III and Cantabria
University (B2017/BMD-3676, AORTASANA-CM, ref. 27/2013;
respectively). Aortas for use as controls were obtained anon-
ymously from multiorgan transplant donors after written
informed consent was obtained from their families. During
preparation of the heart for transplantation, excess ascend-
ing aortic tissue was harvested for the study. Samples from
patients were obtained during elective or emergency aortic
root surgery for aortic aneurysm dissection. Patient clinical
data were retrieved while maintaining anonymity. Tissues
were immediately fixed, kept at room temperature for 48
hours, and embedded in paraffin. DNA and RNA extraction
from paraffin sections were performed with All Prep DNA/
RNA FFPE [formalin-fixed, paraffin-embedded] kit (Qiagen).
Statistical Analysis
Normality of data were checked using a Shapiro–Wilk test.
F-test was used to assess the equality of variance assumption.
Differences between 2 groups were assessed using the
unpaired Student t test, t test with Welch correction for
unequal variances, or Mann–Whitney U test, where appropri-
ate. Differences in experiments with ≥3 groups were analyzed
by 1-way, 2-way, or repeated-measurement 2-way ANOVA,
or a mixed-effects linear model and Newman post hoc test,
as appropriate. For the statistical analysis of RNA-sequencing
data, the P values were corrected using the false discovery
rate method (false discovery rate <0.05). For survival curves,
differences were analyzed with the log-rank (Mantel–Cox)
test. Multiple linear regression analysis adjusted for age was
performed with R software. For all other analysis GraphPad
Prism software 9 was used. Statistical significance was indi-
cated as *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001.
Sample size was chosen empirically based on our previous
experiences in the calculation of experimental variability; no
statistical method was used to predetermine sample size.
Before data analysis, outliers were identified and excluded by
using the Robust Outlier removal method (Q value =5%) to
identify outliers provided with GraphPad Prism 9.
The numbers of animals used are described in the corre-
sponding figure legends. All experiments were done with at
least 3 biologic replicates. Experimental groups were balanced in
terms of animal age, sex, and weight. Animals were genotyped
before experiments, and were all caged together and treated in
the same way. Sex and age are indicated in the figure legends.
Appropriate tests were chosen according to the data distribu-
tion. Variance was comparable between groups in experiments
described throughout the article. For the rest of experiments,
no randomization was used to allocate animals to experimental
groups, and investigators were not blinded to group allocation
during experiments or to outcome assessments.
RESULTS
Aortas From Marfan Syndrome Mice
Present Features of Mitochondrial
Decline
Fbn1C1039G/+ mice reproduce the aortic dilation, aneu-
rysms, and histologic features of aortic medial degenera-
tion found in MFS patients.28 To identify novel molecular
mechanisms underlying TAA formation, we performed
transcriptional analysis in aortas from 24-week-old
Fbn1C1039G/+ mice, showing an intermediate stage of the
aortic disease (Figure1A).34,35 Ingenuity pathway analysis
revealed that 6 of the 10 most altered canonical path-
ways were related to metabolism, including oxidative
phosphorylation, mitochondrial dysfunction, fatty acid
β-oxidation, and the tricarboxylic acid cycle (Figure1B).
Ingenuity pathway analysis prediction of activity of up-
stream regulators identified canonical MFS regulators
such as inducible Nos2 (nitric oxide synthase) and Tgfb1
(transforming growth factor β1).28,32 This analysis pointed
to increased activity of the glycolytic regulator Hif1α (hy-
poxia-inducible factor α) and reduced activity of several
mitochondrial biogenesis and function regulators, includ-
ing Tfam (Figure1C). Fbn1C1039G/+ aortas showed reduced
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
May 25, 2021 Circulation. 2021;143:2091–2109. DOI: 10.1161/CIRCULATIONAHA.120.051171
2096
ORIGINAL RESEARCH
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Figure 1. Mitochondrial function decline in a mouse model of Marfan syndrome.
A through D, RNA-sequencing analysis of aortas from 4 Fbn1C1039G/+ mice with 3 Fbn1+/+ littermates (24-week-old male mice). B, Top 10 significantly changed canoni-
cal pathways predicted by ingenuity pathway analysis based on differentially regulated genes. Metabolism-related pathways are highlighted in blue lettering; P<0.01.
C, Activation or inhibition of upstream regulators predicted by ingenuity pathway analysis (–2>bias-corrected z-score>4; P<0.05). The predicted inhibition of Tfam
is highlighted. D, Expression of genes encoding mitochondrial complexes (Co) and fatty acid oxidation enzymes, and genes related to mitochondrial function and
glycolysis; P < 0.05. E, Quantitative reverse transcription polymerase chain reaction analysis of Tfam mRNA expression and quantitative polymerase chain reaction
analysis of relative mtDNA content in aortic extracts from 20-week-old Fbn1C1039G/+ and Fbn1+/+ male mice. F through I, Primary murine vascular smooth muscle cells
transduced with shFbn1 or shControl for 5 days. F, Quantitative reverse transcription polymerase chain reaction of Hif1a, Pdk1, and representative immunoblots
analysis, and relative quantification of Hif1a and Pdk1 protein levels. G, Quantitative reverse transcription polymerase chain reaction of Tfam and Ppargc1a and
representative immunoblot analysis and quantification of Pgc1α, Tfam, Mt-Nd1, and Mt-Co1. H, Quantitative polymerase chain reaction analysis of relative mtDNA
content in shFbn1- and shControl-transduced vascular smooth muscle cells. I, OCR in shFbn1 and shControl vascular smooth muscle cells at basal respiration and
after addition of the complex V inhibitor oligomycin (I) and fluoro carbonyl cyanide phenylhydrazone (II) to measure maximal respiration, followed by a combina-
tion of rotenone and antimycin A (III). J, Levels of extracellular lactate in the supernatant from shFbn1 and shControl vascular smooth muscle cells. Actin was used
as total protein loading control. Data are mean±SEM Statistical significance was assessed by Student t test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs
Fbn1+/+ mice (E), shControl (F through J). Acaa1b indicates 3-ketoacyl-CoA thiolase B, peroxisomal; Acsl5, Acyl-CoA Synthetase Long Chain Family Member 5; Akt,
serine/threonine kinase 1; Atpaf2, ATP synthase mitochondrial F1 complex assembly factor 2; Ccn2, cellular communication network factor 2; CoI–V, mitochondrial
complexes I-V; Cox8b, cytochrome c oxidase subunit 8B; Cycs, cytochrome c, somatic; Echs1, enoyl-CoA hydratase, short chain 1; Erk, extracellular signal regulated
kinases; Fbn1, fibrillin-1; Hadh, hydroxyacyl-CoA dehydrogenase; Hif1a, hypoxia-inducible factor 1 α; Idh3g, isocitrate dehydrogenase (NAD(+)) 3 non-catalytic sub-
unit gamma; Ivd, isovaleryl-CoA dehydrogenase; Mrtfa, b, myocardin related transcription factor A, B; Mt-Atp6, mitochondrially-encoded ATP synthase membrane
subunit 6; Mt-Co1, 2, mitochondrially encoded cytochrome c oxidase I /II; Mt-Nd1–4I, mitochondrially encoded NADH dehydrogenase 1-4; Myc, Myc protoncogen,
myelocytomatosis oncogene; Ndufa9, 10, NADH:ubiquinone oxidoreductase subunit A9-10; Ndufb8, NADH:ubiquinone oxidoreductase subunit B8; Ndufs4, 8,
NADH:ubiquinone oxidoreductase core subunit S4,8; Ndufv1–3, NADH:ubiquinone oxidoreductase core subunit V1-3; Nos2, nitric oxide synthase 2; (Continued)
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expression of all mitochondrial complex subunits, includ-
ing mitochondria- and nuclear-encoded genes, as well
as of genes related to fatty acid β-oxidation and mito-
chondrial biogenesis and function (Tfam, Ppara [peroxi-
some proliferator-activated receptor α], Pparg [peroxi-
some proliferator-activated receptor γ], Ppargc1a [PPARG
coactivator 1α], Ppargc1b [PPARG coactivator 1β], and
Sod2 [superoxide dismutase 2, mitochondrial]). In con-
trast, genes involved in mitochondrial uncoupling (Ucp2
[uncoupling protein 2]) and glycolytic rewiring (Hif1a [hy-
poxia-inducible factor-1α], Myc) were upregulated (Fig-
ure1D). Quantitative reverse transcription PCR analysis
confirmed reduced expression of Tfam (Figure1E). Tfam
is a nuclear-encoded mitochondrial transcription factor
that controls the transcription, replication, and stabil-
ity of mtDNA.23 Analysis of relative mtDNA content in
Fbn1C1039G/+ aortas revealed below-normal mtDNA con-
tent (Figure1E). To model MFS in vitro, we silenced Fbn1
with lentiviral vectors in primary murine VSMCs (Figure IA
in the Data Supplement). Confirming the transcriptomic
data from MFS mice, shFbn1 VSMCs displayed increased
levels of Hif1α and its targets Pdk1 (pyruvate dehydro-
genase kinase-1) and Slc2a1 (glucose-transporter 1; Fig-
ure 1F), and significantly reduced expression of Pgc1α
(Ppargc1a), as well as Tfam and its targets, MT-Nd1 (mi-
tochondrially-encoded NADH-ubiquinone oxidoreduc-
tase chain 1) and MT-Co1 (Figure1G), correlating with
a reduction in mtDNA content (Figure1H). Flux analysis
of the OCR, an index of mitochondrial oxidative phos-
phorylation, revealed reduced mitochondrial respiration
in Fbn1-silenced cells (Figure1I). This was accompanied
by increased production of extracellular lactate, an indi-
cator of glycolysis (Figure1J). As mitochondrial decline
induces cellular senescence through p53,36 and our inge-
nuity pathway analysis predicts p53 (Tp53) as an active
upstream regulator (Figure1C), we analyzed senescence
features in shFbn1 VSMCs. Both protein and mRNA p53
levels were upregulated in shFbn1 VSMCs (Figure IB in the
Data Supplement) together with increased SABG (senes-
cence-associated β-galactosidase) activity (Figure IC in the
Data Supplement) and increased expression of proinflam-
matory cytokines related to SASPs (senescence-associated
secretory phenotype) such as Tnfa, Il1b (interleukin 1β),
and Il6 (interleukin 6; Figure ID in the Data Supplement).
These data support that aortas from Fbn1C1039G/+
mice or VSMCs carrying MFS mutations present mito-
chondrial respiration decline, rewire their metabolism
toward glycolysis, and display senescence and inflam-
mation features.
Mitochondrial Decline Appears in the
Onset of Aortic Disease in Fbn1C1039G/+ Mice
To assess the stage during the aortic disease in which
mitochondrial decline appears, we performed analysis
of aortic diameter (Figure 2A) and histologic features
(Figure2B and 2C) alongside Tfam and mtDNA levels
(Figure2D and 2E) at different stages of TAA disease in
Fbn1C1039G/+ mice. Aortas from 8-week-old mice already
present significant below-normal Tfam mRNA levels,
mtDNA content, and Mt-Nd1 expression (Figure2D) to-
gether with the appearance of a significant increase in
elastin breaks, proteoglycan deposition, and aortic medial
thickness (Figure2B and 2C). Similarly, some well-known
MFS aortopathy-related genes and metabolic genes al-
ready showed altered levels in aortas from 8-week-old
Fbn1C1039G/+ mice (Figure II in the Data Supplement). In
addition, the diameter of the aorta negatively correlated
with mtDNA levels in MFS mice (Figure2F).
Reduction in Tfam levels induces mtDNA leakage into
the cytoplasm and activates the innate immune pathway
stimulator of interferon genes (cGAS-STING).24,37 Interest-
ingly, both Irf7 (interferon regulatory factor 7) and Isg15
(interferon-stimulated gene 15), which are cGAS-STING
response target genes, were up-regulated in Fbn1C1039G/+
aortas at early stages of the disease (Figure II in the Data
Supplement), supporting a previous report on the role of
this route on aortic degeneration.38 Together these data
suggest that mitochondrial decline appears with the first
signs of aortic remodeling in Fbn1C1039G/+mice.
Decreased Tfam Expression and mtDNA
Levels in Aortas from Human MFS Patients
To assess whether the same metabolic rewiring occurs
in aortas from MFS patients (Figure3A), we measured
TFAM mRNA and mtDNA levels and the expression of
mitochondrial function genes in TAAs from MFS pa-
tients. Compared with healthy control samples, TAA
samples from MFS patients showed significantly lower
levels of mRNA of TFAM and mtDNA (Figure3B), as well
as reduced transcription of genes that encode mitochon-
drial complexes (MT-ND1, SDHA [succinate dehydroge-
nase complex, subunit A], SDHB [succinate dehydroge-
nase complex, subunit B], CYCS [cytochrome complex],
MT-CO1, and MT-ATP6 [mitochondrially-encoded ATP
synthase membrane subunit 6]) or are involved in mito-
chondrial function (PPARA, PPARG, and PPARG1A; Fig-
ure3C and 3D). Moreover, aneurysm samples showed
Figure 5 Continued. OCR, oxygen consumption rate; Mef2d, myocyte enhancer factor 2D; p38-MAPK, p38 mitogen-activated protein kinase; Pccb, propionyl-CoA
carboxylase beta chain, mitochondrial; Pkm, Pyruvate kinase muscle isozyme; Pi3k, phosphatidylinositol 3-kinase; Pparg, Peroxisome proliferator-activated receptor
gamma; Ppara, peroxisome proliferator-activated receptor α: Ppargc1a, b, peroxisome proliferator-activated receptor γ coactivators 1a and 1b, respectively; Rictor,
RPTOR independent companion of MTOR, complex 2; Sdha, d, succinate dehydrogenase complex flavoprotein subunit A, D; shControl, Control Short hairpin RNA;
shFbn1, Short hairpin RNA Fbn1; Sirt1, NAD-dependent deacetylase sirtuin-1; Slc2a1, Glut1, solute carrier family 2 member 1; Sod2, Superoxide dismutase 2, mito-
chondrial; Srf, serum response factor; Suclg1, succinate-CoA ligase GDP/ADP-forming subunit alpha; TCA, tricarboxylic acid cycle; Tfam, mitochondrial transcription
factor A; Tgfb1, transforming growth factor β1; Tp53, tumor protein p53; Uqcr10, Ubiquinol-Cytochrome C Reductase, Complex III Subunit X; Ucp2, mitochondrial
uncoupling protein 2; Uqcrc2, Ubiquinol-Cytochrome C Reductase Core Protein 2; and Uqcrh, Ubiquinol-Cytochrome C Reductase Hinge Protein.
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Figure 2. Decrease of Tfam levels and mitochondrial DNA correlate with aortic deterioration of Fbn1C1039G/+ mice.
Analysis of the progression of mitochondrial and aortic phenotype in Fbn1+/+ and Fbn1C1039G/+ male mice from 4, 8, 12, and 28 weeks old. A, Maximal AsAo and
AbAo diameter. B, Representative images of Van Gienson and Alcian blue histologic staining. C, Quantification of elastin breaks per section and aortic medial
thickness in ascending aortas at the indicated ages. D, Relative quantitative reverse transcription polymerase chain reaction analysis of Tfam and Mt-Co1 mRNA
expression in aortic extracts from Fbn1+/+. E, Quantitative polymerase chain reaction analysis of relative mtDNA content in aortas from 4-week-old Fbn1+/+ mice
and (F) multiple linear regression adjusted for age, between the AsAo diameter and mtDNA/nDNA levels; adjusted R2 and P value are indicated. Histograms show
mean±SEM. Aortic diameter is presented in box and whisker plots showing maximal and minimal values and 75th and 25th percentiles. Statistical significance
was assessed by 2-way ANOVA (A, C, E), Student t test (D), and multiple linear regression (F). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. Fbn1+/+ mice;
##P<0.01, ###P<0.001 vs. Fbn1C1039G/+ mice. AbAo indicates abdominal aorta; AsAo, ascending aorta; Fbn1, fibrillin-1; mtNd1, mitochondrially-encoded NADH
ubiquinone oxidoreductase core subunit 1; n.s., not significant; and Tfam, mitochondrial transcription factor A.
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
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Figure 3. Decrease of TFAM levels and mitochondrial respiration in samples from MFS patients.
A, Analysis of human ascending aortic samples from MFS patients and control donors. B, Quantitative reverse transcription polymerase chain reaction analysis of
TFAM mRNA and quantitative polymerase chain reaction analysis of relative mtDNA content. C, Expression of genes encoding mitochondrial complex components
and (D) genes related to mitochondrial function and glycolysis. E, Representative medial layer sections of immunohistochemical analysis of MT-ND1 (CoI), SDHA
(CoII), TFAM, HIF1A, and MYC levels and (F) quantification. G through K, Primary fibroblasts from 4 patients with MFS and 4 healthy controls. G, Quantitative poly-
merase chain reaction analysis of mtDNA content and quantitative reverse transcription polymerase chain reaction analysis of TFAM and PPARGC1A expression (H)
and representative TFAM and PGC1α immunoblotting (n=4). I, mRNA of MT-ND1, SDHA, CYCS, MT-CO1, MT-ATP6, (J) UCP2, HIF1A, and MYC as assessed by quan-
titative reverse transcription polymerase chain reaction , in extracts from human MFS and Control fibroblast. K, OCR after addition of oligomycin (I), fluoro carbonyl
cyanide phenylhydrazone (II), and a combination of rotenone and antimycin-A (III) and extracellular lactate levels. Data are mean±SEM. Statistical significance was
assessed by Student t test *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Control. ACTIN indicates beta actin; COI–V, mitochondrial complexes I-V; CYCS, Cy-
tochrome C, Somatic; HIF1A, hypoxia-inducible factor 1 α; MFS, Marfan syndrome; MT-ATP6, mitochondrially-encoded ATP synthase membrane subunit 6; MT-CO1,
mitochondrially-encoded cytochrome c oxidase I; MT-ND1, mitochondrially-encoded NADH ubiquinone oxidoreductase core subunit 1; MYC, MYC proto-oncogene,
bHLH transcription factor; PPARA, Peroxisome proliferator-activated receptor alpha; PPARAGC, peroxisome proliferator-activated receptor γ coactivator; PPARAGC1a,
Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; PGC1α, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (protein);
SDHA, B, succinate dehydrogenase complex flavoprotein subunit A-B; TFAM, mitochondrial transcription factor A; and UCP2, mitochondrial uncoupling protein 2.
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
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upregulation of genes involved in mitochondrial uncou-
pling and glycolytic rewiring (UCP2, HIF1A, and MYC;
Figure3D). Histologic analysis of TFAM, the mitochon-
drial proteins MT-ND1, SDHA and the glycolysis-related
genes HIF1A and MYC supported glycolytic rewiring in
MFS aortic samples (Figure3E and 3F). Coimmunostain-
ing for smooth muscle actin confirmed that mitochondri-
al gene down-regulation was specific to VSMCs (Figure
III in the Data Supplement). Moreover, we used primary
skin fibroblasts from 4 individuals with MFS and 4 healthy
donors to analyze the mitochondrial respiratory capacity
in vitro. MFS fibroblast presented a reduction in the total
mtDNA content, TFAM and PPARGC1a mRNA, and pro-
tein expression (Figure3G and 3H). In line with human
MFS aortas, primary MFS skin fibroblasts showed a de-
crease of the mRNA levels of MT-ND1, SDHA, MT-CO1,
and MT-AP6 and increased expression of UCP2, HIF1A,
and MYC (Figure3I and 3J). Moreover, MFS fibroblasts
showed a reduction in the OCR and increased lactate
production (Figure3K). Altogether, these data support
that aortas from MFS present metabolic rewiring toward
glycolysis because of mitochondrial function decline.
Extracellular Regulation of Mitochondrial
Respiration in MFS
Because elevated arterial stiffness is a characteristic
of TAA,16,17,39 and recent evidence supports that ECM
stiffness and composition affect cellular metabolism,22
we hypothesized that VSMC metabolism could be
regulated by the microenvironment. To investigate this
hypothesis, we transferred control VSMCs to plates
coated with ECM-produced by Fbn1-deficient VSMCs
(Figure4A). Importantly, ECM from TAA cells induced a
reduction in the OCR and increased lactate production
in control VSMCs (Figure4B). In addition, TAA ECM in-
duced a decrease in the levels of Tfam mRNA and in
the mtDNA content and reduced the transcription of
the mitochondrial genes Mt-Co1 and Ppargc1a while
increasing the transcription of the glycolytic transcrip-
tion factor Hif1a (Figure4C). Moreover, control VSMCs
cultured on ECM produced by Fbn1-deficient VSMCs
up-regulated the expression of synthetic genes (Tgfb1,
Spp1 [osteopontin], andCcn2 [cellular communication
network factor 2]; Figure4D), suggesting that both the
metabolism and the phenotype of VSMCs depends on
signals coming from the extracellular matrix.
Mitochondrial Dysfunction in VSMCs
Causes Aortic Aneurysm
To investigate whether the mitochondrial decline plays
a role in VSMC function and in the progression of aor-
tic diseases, we induced mitochondrial dysfunction in
VSMCs by targeting Tfam. For in vitro experiments, we
transduced aortic VSMCs from Tfamflox/flox mice with
lentiviral vectors encoding the Cre recombinase. Tfam
deletion was confirmed by analyzing Tfam mRNA and
protein levels (Figure5A). In accordance with its role in
regulating mtDNA levels, Tfam deletion in VSMCs led
Figure 4. ECM derived from thoracic aortic aneurysm cells decreases Tfam levels and mitochondrial respiration.
A, Primary vascular smooth muscle cells transduced with shFbn1 or shControl were cultured for 5 days, lead to produce ECM, then the matrices were decel-
lularized and shControl cells were seeded. B, OCR in shControl vascular smooth muscle cells seeded in shControl and ShFbn1-ECM at basal respiration and after
addition of oligomycin (I) and fluoro carbonyl cyanide phenylhydrazone (II) to measure maximal respiration, followed by a combination of rotenone and antimycin
A (III) and extracellular lactate levels. C, Quantitative reverse transcription polymerase chain reaction analysis of Tfam, Mt-co1, Ppargc1, and Hif1a, and quantita-
tive polymerase chain reaction analysis of mtDNA content. D, Quantitative reverse transcription polymerase chain reaction analysis of synthetic genes Tgfb1,
Ccn2, and Spp1. Statistical significance was assessed by Student t test. *P<0.05, **P<0.01 vs Control. Ccn2 indicates cellular communication network factor 2;
ECM, extracellular matrix; Hif1a, hypoxia-inducible factor 1 α; mt-Co1, mitochondrially-encoded cytochrome c oxidase I; OCR, oxygen consumption rate; Ppargc1,
peroxisome proliferator-activated receptor γ coactivator 1; shFbn1, Short hairpin RNA Fbn1; Spp1, secreted phosphoprotein 1; Tfam, mitochondrial transcription
factor A; and Tgfb1, transforming growth factor β1;
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
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ORIGINAL RESEARCH
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to a reduction in mtDNA content that correlated with a
decreased expression of the mtDNA-encoded genes Mt-
Co1 and Mt-Nd1 and a reduced OCR, alongside with
an increase in Slc2a1 expression and lactate production
(Figure5A–5D). We also observed a sharp decrease in
the mRNA expression of genes involved in contractility,
such as smooth muscle Myh11, Acta2, Tagln ( transge-
lin), Cnn1 (calponin), and Smtn (smoothelin; Figure5E).
Moreover, this was accompanied by increased mRNA
expression of genes related to the secretory pheno-
type, such as Spp1, the metalloproteinases Mmp2
and Mmp9, and the inducible Nos2 (Figure 5F). The
increased Mmp9 expression was accompanied by a
marked increase of Mmp9 enzymatic activity in cell su-
pernatants (Figure5F). As observed in Fbn-1–deficient
VSMCs, Tfam-depleted VSMCs showed an increase of
p53 expression, senescent-associated β-galactosidase
activity, and the proinflammatory cytokines Tnfa and Il6
mRNA levels (Figure IV in the Data Supplement).
To analyze the effect of Tfam deletion in VSMCs in
vivo, we crossed Tfamflox/flox mice23 with mice express-
ing the CreERT2 fusion protein under the smooth muscle
myosin (Myh11) promoter (Myh11-CreERT229; Figure5G).
Analysis 28 weeks after tamoxifen treatment confirmed
efficient abrogation of Tfam expression in aortas from
Myh11-CreERT2Tfamflox/flox mice, but not in tamoxifen–
treated Myh11-CreERT2TfamWt/Wt (hereafter referred to
as SM-Tfam−/− and SM-Tfam+/+ mice, respectively; Fig-
ure5H). Tfam deletion was accompanied by a decrease
in mtDNA content (Figure5H). Lifespan analysis showed
a significant decrease in survival rate, with 100% of
SM-Tfam−/− mice dying before 33 weeks after tamoxi-
fen (Figure5I). Longitudinal analysis of vascular pheno-
type revealed a decrease in blood pressure alongside
with an increase in aortic diameter (Figure5J and 5K).
Histologic analysis of SM-Tfam−/− mice 28 weeks after
tamoxifen revealed aortic dissections, intramural hema-
tomas, and medial degeneration, with elastin lamina
degradation and proteoglycan accumulation in the as-
cending aorta (Figure5L). Aortas from SM-Tfam−/− mice
also showed defective vascular contractility responses
to high K+ solution, the α1-adrenergic agonist phen-
ylephrine, and the thromboxane receptor A2 agonist
U46619 (Figure5M). Similar to MFS, aortas from SM-
Tfam−/− mice showed an increase of the cGAS-STING
pathway genes (Irf7, Isg15, Tmem173 [transmembrane
protein 173], Mb21d1 [mab-21 domain-containing
protein 1]), proinflammatory cytokines, such as Il1b,
Tnfa, and Il6, and the senescent marker p21 (Cdkn1a;
Figure IVC in the Data Supplement). Consistently, Tfam
deficiency in VSMCs greatly affects the vascular con-
tractility and function.
To test how SM-Tfam−/− mice respond to a hyperten-
sive challenge, we infused SM-Tfam+/+ and SM-Tfam−/−
mice with Ang II 56 days after tamoxifen (Figure6A).
Analysis of blood pressure showed a modest increase
in SM-Tfam−/− mice (Figure 6B) compared with SM-
Tfam+/+. Ultrasonography revealed a rapid increase in
the diameter of the ascending and abdominal aortas
of SM-Tfam −/− mice, supporting the predisposition of
these mice to development of aortic aneurysms (Fig-
ure6B–6E). Most importantly, treatment of SM-Tfam−/−
mice with Ang II triggered aortic aneurysms and lethal
aortic dissections, reducing mean survival (Figure6E).
Postmortem analysis revealed the presence of intramu-
ral hematomas and aortic ruptures with hemothorax or
hemoabdomen (Figure6F). Histologic analysis of tho-
racic and abdominal aortic sections showed increased
aortic diameter, aortic dissections, intramural hema-
tomas, false lumen formation, and features of medial
degeneration, including elastic fiber fragmentation
and disorganization, medial thickening, and accumula-
tion of proteoglycans (Figure6G). These data support
that mitochondrial function in VSMCs is an important
regulator of aortic function, and demonstrate that mi-
tochondrial dysfunction in VSMCs induces aortic aneu-
rysm and lethal dissections.
NR Normalizes Mitochondrial Respiration
in VSMCs Carrying TAA Mutations
NAD+ is a cofactor for several enzymes with a critical
role in the maintenance of cellular metabolism and
mitochondrial function.40 NAD+ precursor supplemen-
tation has been proposed as a strategy to improve mi-
tochondrial function conditions related to mitochon-
drial decline,41–43 including senescence.44 NR is a NAD
precursor that improves mitochondrial metabolism by
increasing Pgc1α and Tfam expression through sirtuin
activity.42,45 Importantly, cellular production of NAD+ via
Nampt (nicotinamide phosphoribosyltransferase) pro-
tects against DNA damage and premature senescence
in VSMCs.46 To investigate the therapeutic potential of
NAD+ boosting mitochondrial metabolism in familial
aortic aneurysms, we treated shFbn1 VSMCs with NR.
Exposure of shFbn1 VSMCs to NR for 5 days increased
the expression of Pparg1a and Tfam, correlating with
increased mtDNA content and the expression of the
mtDNA-encoded Mt-Co1 transcript (Figure7A and 7B).
NR increased OCR and decreased lactate production in
shFbn1 VSMCs to levels observed in ShControl VSMCs
(Figure7C and 7D). NR treatment of shFbn1 VSMCs
decreased the expression and activity of the proremod-
eling matrix metalloproteinases Mmp9 and Mmp2 and
the profibrotic genes Spp1 and Col1a1 (collagen, type
1, α1; Figure7E and 7F).
To confirm the potential of boosting mitochondri-
al metabolism to revert MFS features in humans, we
performed in vitro analysis in primary skin fibroblast
cultures from 4 healthy donors and 4 MFS patients.
NR treatment restored the mRNA levels of TFAM and
mtDNA (Figure7G). NR increased the mRNA levels of
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
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Figure 5. Ablation of Tfam in vascular smooth muscle cells induces synthetic phenotype and aortic remodeling.
A through F, Primary mouse Tfamflox/flox vascular smooth muscle cells were transduced with LV-Mock or LV-Cre lentivectors and analyzed after 10 days. A, Quantita-
tive reverse transcription polymerase chain reaction analysis of relative Tfam, and RT Mt-Nd1, Mt-Co1, and Slc2a1 mRNA expression and representative immunoblot
analysis of Tfam and Mt-Co1; Vdac and Tub were used as mitochondrial and total protein loading controls, respectively. B, Quantitative polymerase chain reaction
analysis of relative mtDNA content. C, OCR at (D) basal respiration and after addition of oligomycin (I) and fluoro carbonyl cyanide phenylhydrazone (II) to measure
maximal respiration, followed by a combination of rotenone and antimycin A (III); and normalized extracellular lactate levels. E, Quantitative reverse transcription
polymerase chain reaction assessed relative mRNA expression of the smooth muscle contractile genes Myh11, Acta2, Cnn1, Tagln, and Smtn. F, Quantitative reverse
transcription polymerase chain reaction assessed relative mRNA expression of the vascular smooth muscle cells synthetic phenotype genes Spp1, Nos2, Mmp9, and
Mmp2. F, right, Representative gelatin zymograph from 24 h conditioned medium, indicating Mmp9 and Mmp2 enzymatic activity. (Continued )
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
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MT-CO1, MT-ND6, and HIF1A (Figure7G and 7H), re-
stored OCR, and decreased lactate production in prima-
ry skin fibroblasts from MFS patients (Figure7I and 7J).
Furthermore, NR reduced the expression of the ECM
genes COL1A1 and ACAN (aggrecan) and the profibrot-
ic factors TGFB3 and CCN2 (connective tissue growth
factor; Figure7K). These data indicate that incubation
with NR restores TFAM levels and improves mitochon-
drial respiration in vitro in cells from MFS patients.
NR Treatment Reverts Aortic Aneurysm in
a Mouse Model of MFS
We next tested the therapeutic potential of NR to pre-
vent or even revert the development of aneurysm in
MFS mice by modulating mitochondrial metabolism.
Starting at 20 weeks old, Fbn1C1039G/+ mice received
IP injections of NR every second day for 28 days (Fig-
ure8A; Figure VA in the Data Supplement). NR restored
aortic Tfam and Mt-Co1 mRNA expression and mtDNA
content to normal levels in MFS mice (Figure8B). Im-
portantly, aortic dilation and BP were completely nor-
malized after 7 days of treatment in both male and
female mice (Figure8C and 8D; Figure VB in the Data
Supplement). Moreover, NR restored histologic features
of aortic degeneration in MFS, such as medial thicken-
ing, elastic fiber fragmentation, proteoglycan deposi-
tion, and actin polymerization (Figure8E; Figure VC in
the Data Supplement).
To characterize the effect of NR treatment at the mo-
lecular level, we performed RNA-sequencing on aortas
from MFS and control mice. Hierarchical clustering of
the transcriptomic analysis revealed that NR reverted
the transcriptional changes observed in Fbn1C1039G/+ aor-
tas, bringing expression levels closer to controls than to
Fbn1C1039G/+ mice (Figure8F). NR increased the expres-
sion levels of genes related to mitochondrial complexes,
fatty acid β-oxidation, and mitochondrial function, in-
cluding Tfam and Ppargc1a, and reduced the expression
of genes related to glycolytic rewiring, such as Hif1a and
Myc (Figure8F). Notably, NR also decreased the expres-
sion of classical MFS-affected genes such as transcripts
involved in the TGFβ pathway and those encoding
ECM-related proteins (Figure 8G). Additionally, NR
brought the expression of some transcription factors
involved in the maintenance of the contractile pheno-
type and genes related to the smooth muscle contrac-
tion apparatus, such as smooth muscle Acta2 and Cnn1,
down to Fbn1+/+ (Figure8H). Thus, boosting mitochon-
drial function with NR rapidly restores the transcriptional
signature in aortas from MFS mice and fully reverts the
associated aortic wall remodeling, aortic dilation, and
medial degeneration. Taken together, our data highlight
VSMC metabolism as a critical mediator of hereditable
TAAs and suggest that the use of mitochondrial-boost-
ing compounds is a promising pharmacologic strategy
for treating patients with these hereditary disorders.
DISCUSSION
We found that aortas from Fbn1C1039G/+ mice and hu-
man MFS patients present low TFAM expression, below-
normal mtDNA levels, and a decline in mitochondrial
respiration. This decline in oxidative phosphorylation
was compensated by increased glycolytic metabolism
and appears at the onset of the aortic disease. In line
with our data, aortas from the Cutis laxa mouse model
showed decreased mitochondrial respiration and in-
creased glycolysis,47 suggesting that mitochondrial de-
cline could be a common driver and hallmark of differ-
ent hereditary TAAs.
Next, we tried to elucidate how aneurysms caused by
mutations related to ECM and VSMC contractile function
converge on the impairment of TFAM expression and mi-
tochondrial dysfunction. Recent reports in epithelial cells
show that stiff matrices produce a mechanical regulation
of glycolysis via remodeling of the cytoskeleton architec-
ture.22 Because VSMCs and aortas carrying TAA mutations
show an increase of ECM deposition, active peptides and
growth factors, stiffness, and mechanotransduction,17,18,48
we hypothesized that the ECM could drive this metabolic
rewiring toward glycolysis. In vitro experiments in VSMCs
seeded in matrices produced by Fbn1-deficient VSMCs
support that metabolism is controlled by the ECM. Hence,
changes in the composition of the ECM during aneurysm
development drives metabolic rewiring toward glycolysis.
Figure 5 Continued. G, Experimental design for H through L: SM-Tfam+/+ and SM-Tfam−/− mice were treated with tamoxifen at 5 weeks old. H, Quantitative reverse
transcription polymerase chain reaction analysis of Tfam expression and quantitative polymerase chain reaction analysis of mtDNA content in aortic extracts from SM-
Tfam+/+ and SM-Tfam−/− mice 28 weeks after tamoxifen injections. I, Percent survival after tamoxifen injections in SM-Tfam+/+ and SM-Tfam−/− mice; n=20. J, Evolution
of systolic and diastolic blood pressure after tamoxifen injections in SM-Tfam+/+ and SM-Tfam−/− mice. K, Evolution of maximal ascending aorta and abdominal aorta
diameters after tamoxifen treatment in SM-Tfam+/+ and SM-Tfam−/− mice. L, Representative images of histologic staining with H/E, EVG, and Alcian blue in ascending
aortas from SM-Tfam+/+ and SM-Tfam−/− mice 28 weeks after tamoxifen injection. Black arrowheads indicate aortic dissections; red arrowheads indicate intramural
hematomas; n=4. M, Aortic contractile responses to high KCl solution and concentration–response curves to Phe and U46619; n=5. Data are mean±SEM. Statistical
significance was assessed by Student t test (A through H), log-rank (Mantel–Cox) test (I), mixed-effects linear model (J, K), or 2-way ANOVA repeated measures (M,
right). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs Lv-Mock (A through F) or vs SM-Tfam+/+ mice (H through M). AbAo indicates abdominal aorta; Acta2,
actin α2, smooth muscle; AsAo, ascending aorta; Bp, blood pressure; Cnn1, calponin 1; Cre, Cre recombinase; EVG, elastin Van Gienson; H/E, hematoxylin-eosin;
LV-Cre, Cre-expressing; LV-Mock, green fluorescent protein–expressing; Mmp2, 9, matrix metalloproteinase 2 and 9, respectively; Mt-Co1, mitochondrially-encoded cy-
tochrome c oxidase I; Mt-Nd1, mitochondrially-encoded NADH ubiquinone oxidoreductase core subunit 1; Myh11, myosin heavy chain 11; Nos2, nitric oxide synthase
2; OCR, oxygen consumption rate; Phe, phenylephrine; Slc2a1, solute carrier family 2 member 1; SM-Tfam+/+, tamoxifen–treated Myh11-CreERT2TfamWt/Wt mice; SM-
Tfam−/−, tamoxifen–treated Myh11-CreERT2Tfamflox/flox mice; Smtn, smoothelin; Spp1, secreted phosphoprotein 1; Tagln, transgelin; Tfam, mitochondrial transcription
factor A; Tmx, tamoxifen; Tub, tubulin; U46619, U46619 synthetic analog of the prostaglandin PGH2; and Vdac, Voltage-dependent anion channel mitochondrial.
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Figure 6. Infusion of Ang II in SM-Tfam−/− mice predisposes mice to lethal aortic aneurysm and dissections.
A, Experimental design: 56 days (8 weeks) after tamoxifen injections, Ang II minipumps were implanted in 6 SM-Tfam+/+ and 10 SM-Tfam−/− male mice. Ultrasound
and blood pressure (BP) analysis was performed 6 times (empty triangles). B, Evolution of systolic and diastolic BP (top) and maximal AsAo and AbAo diameters
(bottom) on Ang II infusion. C, Representative aortic ultrasound images after 28 days of Ang II infusion in SM-Tfam+/+ and SM-Tfam−/− mice. Discontinuous red lines
mark the lumen boundary and discontinuous yellow lines and arrows denote the lumen diameter. D, Representative macroscopic images of aortas from the same
animal cohort shown in A. Red arrowheads indicate aneurysms, dissections, and intramural hematomas. E, Aortic aneurysm incidence and percent survival of Ang
II−infused SM-Tfam+/+ and SM-Tfam−/− mice from the same cohort shown in A. F, Incidence and localization of lethal aortic dissections and IMH in the same cohort
shown in A. G, Representative histologic analysis on sections of AsAo, TDAo, and AbAo from the same cohort shown in A. Statistical significance was assessed
by mixed-effects linear model (B) and log-rank (Mantel–Cox) test (E). Data are mean±SEM. Red arrows indicate intramural hematomas. *False lumen; P<0.05,
**P<0.01, ***P<0.001, ****P<0.0001 vs. SM-Tfam+/+ mice. AbAo indicates abdominal aorta; Alcian B, Alcian blue; AngII, angiotensin II; AsAo, ascending aorta;
BP, blood pressure; EVG, elastin Van Gienson; H/E, hematoxylin eosin; IMH, intramural hematomas; Masson T, Masson’s trichrome; MFS, Marfan syndrome; NR,
nicotinamide riboside; SM-Tfam+/+, tamoxifen–treated Myh11-CreERT2TfamWt/Wt mice; SM-Tfam−/−, tamoxifen–treated Myh11-CreERT2Tfamflox/flox mice; TDAo, thoracic
descending aorta; Tfam, mitochondrial transcription factor A; Tmx, tamoxifen; TorAo, Thoracic aorta; and TorAo/AbAo, Thoracic/Abdominal aorta.
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Figure 7. NR increases Tfam levels and mitochondrial respiration in murine and human MFS cells.
A through F, Primary vascular smooth muscle cells transduced with shFbn1 or shControl were treated with NR for 5 days. A, Quantitative reverse transcription
polymerase chain reaction (RT-qPCR) analysis of Tfam and quantitative polymerase chain reaction analysis of mtDNA content. B, RT-qPCR analysis of Ppargc1a
and Mt-Co1 mRNA expression. C through D, OCR in shFbn1 and shControl vascular smooth muscle cells after incubation with or without NR at basal respiration,
and after addition of oligomycin (I) and fluoro carbonyl cyanide phenylhydrazone (II) to measure maximal respiration, followed by a combination of rotenone
and antimycin A (III) and extracellular lactate levels. E, Representative gelatin zymogram images (left) and enzymatic activity analysis of Mmp2 and Mmp9 in 24h
conditional medium (right). F, RT-qPCR analysis of Mmp9, Mmp2, Spp1, and Col1a1 mRNA expression. G through K, Effect of NR on primary dermal fibroblasts
from 4 MFS patients and 4 healthy donors (Control); cells were treated with NR for 5 days. G, RT-qPCR analysis of TFAM mRNA expression, quantitative polymerase
chain reaction analysis of mtDNA content, and RT-qPCR analysis of MT-CO1, MT-ND6, TFAM, and (H) HIF1A mRNA expression. I, OCR after addition of oligomycin
(I), fluoro carbonyl cyanide phenylhydrazone (II), and a combination of rotenone and antimycin A (III). J, Basal and maximal respiration rate and extracellular lactate
levels. K, RT-qPCR analysis of COL1A1, ACAN, CCN2, and TGFB3 mRNA expression. Data are mean±SEM. Statistical significance was assessed by 1-way ANOVA:
*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs ShControl or Control; # P<0.05, ## P<0.01, ###P<0.001, ####P<0.0001 vs ShFbn1 or MFS NR. ACAN indi-
cates aggrecan; CCN2, cellular communication network factor 2; COL1A1, collagen type I α1 chain; HIF1A, hypoxia-inducible factor 1 α; MFS, Marfan syndrome;
Mmp2, 9, matrix metalloproteinases 2 and 9, respectively; MT-CO1, mitochondrially-encoded cytochrome c oxidase I; MT-ND6, mitochondrially-encoded NADH-
ubiquinone oxidoreductase chain 6; NR, nicotinamide riboside; OCR, oxygen consumption rate; Ppargc1a, peroxisome proliferator-activated receptor γ coactivator
1a; Spp1, secreted phosphoprotein 1; TFAM, mitochondrial transcription factor A; and TGFB3, transforming growth factor β3.
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Figure 8. Nicotinamide riboside treatment restores aortic homeostasis in a mouse model of Marfan syndrome.
A, Experimental design: 20-week-old Fbn1+/+ and Fbn1C1039G/+ male mice were treated with NR or vehicle for 28 days (blue arrowheads). Ultrasound and BP analysis
was performed 5 times (empty triangles); n=9. B, Quantitative reverse transcription polymerase chain reaction analysis of Tfam, quantitative polymerase chain reac-
tion analysis of relative mtDNA content, and mt-Co1 mRNA. C, Representative aortic ultrasound images after 28 days of vehicle or NR treatment. Discontinuous red
lines mark the lumen boundary, and discontinuous yellow lines and arrows denote the lumen diameter. D, Evolution of maximal AsAo and AbAo diameter (top)
and systolic and diastolic BP (bottom) on NR treatment; n=9. E, Representative histologic staining with EVG and Alcian blue in the AsAo (top) and with EVG in the
AbAo (middle) and confocal imaging of F-actin (red), elastin (green, autofluorescence), and 4′,6-diamidino-2-phenylindole–stained nuclei (blue) in the descending
thoracic aorta (bottom); and quantification of elastin breaks and aortic medial thickness. F through H, RNA-sequencing analysis of aortic medial tissue from Fbn1+/+
mice (n=3) and from Fbn1C1039G/+ mice treated for 28 days with vehicle (n=4) or NR (n=4). F, Hierarchical clustering showing the top 200 most significant differentially
expressed genes (by adjusted P < 0.05) between the 3 groups of mice (left) and gene expression heatmap for mitochondrial complex, (Continued )
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
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In epithelial cells, PI3K (phosphoinositide 3-kinase) signal-
ing coordinates glycolytic metabolism and actin remodel-
ing. Activation of PI3K mobilizes aldolase from F-actin,
increasing glycolysis.20,49 In endothelial cells, another gly-
colytic pathway enzyme, PFKFB (phospho-fructokinase 2/
fructose-2,6 bisphosphatase), regulates vessel sprouting
through the coordination of glycolytic metabolism and
the actin cytoskeleton.50 In dermal fibroblasts, downregu-
lation of fatty acid β-oxidation and an increased glycoly-
sis rate contribute to ECM alterations, promoting fibro-
sis.51 In VSMCs from pulmonary hypertension patients,
inhibiting glycolysis by silencing α-enolase prevents the
malignant secretory phenotype.52 Altogether, these data
support that there is a bidirectional connection between
the ECM composition and the cellular metabolism with a
critical implication in aortic tissue homeostasis (Figure VI
in the Data Supplement)
To demonstrate the importance of VSMC metabolism
in the development of aneurysms, we generated a con-
ditional mouse model with specific deletion of Tfam in
VSMCs. Tfam deficiency in VSMC promotes mitochondri-
al dysfunction and a metabolic rewiring toward glycolysis.
Tfam-depleted VSMCs acquire a senescent and proinflam-
matory phenotype together with an impairment in their
contractile function. Moreover, these mice develop aortic
aneurysm, medial degeneration, and lethal dissections.
Similarly, silencing of Tfam in fibroblast prevents differ-
entiation toward myofibroblasts and the acquisition of a
contractile phenotype.53 During the last decade, it became
evident that signals from stressed mitochondria regulate
senescence and inflammatory cascades in multiple cell
types.36,37,54 The release of mtDNA from the mitochondria
to the cytosol activates the cGAS-STING response and
contributes to the progression of the aneurysm.37,38 In
the same line, we have observed the activation of cGAS-
STING route in both in aortas from Fbn1C1039G/+ mice and
in SM-Tfam−/− mice, further confirming the involvement of
this route in the pathogenesis of the aortic diseases.
Besides the high incidence and mortality risk of rup-
tured aneurysms in inherited TAA patients, there are still
limited therapeutic options to delay TAA progression to
dissection and none to prevent it. Current pharmacologic
standard treatments for TAA are based on BP control with
β-adrenergic blockers or the Ang II receptor-1 antagonist
losartan, which slow down aortic dilation but do not pre-
vent dissection.55,56 Therefore, the current management
of aortic aneurysms relies on surgical prophylactic repair,
which shows significantly higher perioperative mortality
and morbidity.57 Consequently, it is imperative to identify
novel molecular mediators involved in hereditable TAA
pathophysiology to design new pharmacologic strate-
gies. Since the use of NAD+ precursors has emerged as
an effective approach to boost mitochondrial dysfunc-
tion in different pathologies,40,41,45 we investigated the
pharmacologic potential of NR in TAAs associated with
genetic disorders. We observed that NR rapidly raised
Tfam levels, improved mitochondrial metabolism, and
normalized aortic function and diameter in the Marfan
mouse model of TAA. Our results indicate that glycolytic
metabolism is a common driver of hereditary TAAs and
NR could be trialed for the treatment of different types of
genetic disorders that cause TAA. Further research would
be required to determine if this approach could also be
effective against other vasculopathies, such as cerebral
aneurysm or pulmonary hypertension.
ARTICLE INFORMATION
Received August 28, 2020; accepted February 26, 2021.
The Data Supplement is available with this article at https://www.ahajournals.
org/doi/suppl/10.1161/circulationaha.120.051171.
Correspondence
María Mittelbrunn, PhD, Immunometabolism and Inflammation Laboratory,
Centro de Biología molecular Severo Ochoa, C/ Nicolás Cabrera 1, 28049 Ma-
drid, Spain. Email mmittelbrunn@cbm.csic.es
Affiliations
Departamento de Biología Molecular, Centro de Biología Molecular Severo
Ochoa, Consejo Superior de Investigaciones Científicas Universidad Autónoma
de Madrid, Spain (J.O., E.G-R., G.D-M., J.F.A., E.M.B., P.A., M.M.). Instituto de
Investigación Sanitaria del Hospital 12 de Octubre (i+12), Madrid, Spain (J.O.,
Figure 8 Continued. fatty acid β-oxidation, mitochondrial function, and proglycolytic genes (right). G, Expression heatmap for genes encoding extracellular matrix–related
proteins and (H) smooth muscle contractile apparatus. The heatmap was obtained from DESeq2 analysis. Statistical significance was assessed by 1-way ANOVA (B, E) or
2-way repeated measurements ANOVA (D). **P<0.01, ***P<0.001, ****P<0.0001 for Fbn1C1039G/+ vs Fbn1+/+; #P<0.05, ##P<0.01, ####P<0.0001 for Fbn1C1039G/+NR vs
Fbn1C1039G/+. AbAo indicates abdominal aorta; Acaa1b, acetyl-Coenzyme A acyltransferase 1B; Acadm, acyl-CoA dehydrogenase medium chain; ACAN, aggrecan; Acsl5,
Acyl-CoA Synthetase Long Chain Family Member 5; Acox1, acyl-Coenzyme A oxidase 1, palmitoyl; Acta2, actin, alpha 2, smooth muscle; AsAo, ascending aorta; Bmp 2,
bone morphogenetic protein 2; BP, blood pressure; Ccn2, cellular communication network factor 2; Col1a1, collagen type I α1 chain; Cox8b, cytochrome c oxidase subunit
8B; Cnn1, calponin 1; Cpt1a, carnitine palmitoyltransferase 1A; Cycs, cytochrome c, somatic; DAPI, 4′,6-diamidino-2-phenylindole; Echs1, enoyl-CoA hydratase, short chain
1; EVG, elastin Van Gienson; F-Actin, filamentous actin; Fbn1, fibrillin-1; Fgf2, fibroblast growth factor 2; Fn1, Fibronectin1; Hadh, hydroxyacyl-CoA dehydrogenase; Hif1a,
hypoxia-inducible factor 1 α; Itga5, integrin subunit alpha 5; Ivd, isovaleryl-CoA dehydrogenase; Klf15, Kruppel-like factor 15; Lrpprc, leucine rich pentatricopeptide repeat
containing; Mmp3, matrix metalloproteinase 3; Mrtfa, b, myocardin related transcription factor A,B; Mt-Atp6, mitochondrially-encoded ATP synthase membrane subunit 6;
Mt-Co1, 2, mitochondrially encoded cytochrome c oxidase I,II; Mt-Nd1–4l, mitochondrially-encoded NADH-ubiquinone oxidoreductase chains 1–4l, respectively; Myc, MYC
proto-oncogene, bHLH transcription factor; Myh11, myosin heavy chain 11; Mylk, myosin light chain kinase; Myocd, myocardin; Ndufa9, 10, NADH:ubiquinone oxidoreduc-
tase subunit A9, 10; Ndufb8, NADH:ubiquinone oxidoreductase subunit B8; Ndufs4, 8, NADH:ubiquinone oxidoreductase core subunit S4; Ndufv1–3, NADH:ubiquinone
oxidoreductase core subunit V1, V3; NR, nicotinamide riboside; Pdgfa, platelet derived growth factor subunit A; Pparg, Peroxisome proliferator-activated receptor gamma;
Ppara, peroxisome proliferator-activated receptor α; Ppargc1a, b, peroxisome proliferator activated receptor γ coactivators 1a and b, respectively; Ppp1r12a, protein phos-
phatase 1 regulatory subunit 12A; Sdc4, Syndecan4; Sdha, d, succinate dehydrogenase complexes A and D, respectively; Serpine1, serpin family E member 1; Slc2a1, solute
carrier family 2 member 1; Spp1, secreted phosphoprotein 1; Sod2, superoxide dismutase 2, mitochondrial; Srf, serum response factor; Tagln, transgelin; Tfam, mitochondri-
al transcription factor A; Tgfb1–3, transforming growth factors β1–3, respectively; Thbs1, thrombospondin 1; Uqcr10, ubiquinol-cytochrome c reductase, complex III subunit
X; Uqcrc2, ubiquinol-cytochrome c reductase core protein 2; Uqcrh, ubiquinol-cytochrome c reductase hinge protein; and Vcan, Versican.
Oller et al Aberrant Glycolysis in Familial Aortic Aneurysms
May 25, 2021 Circulation. 2021;143:2091–2109. DOI: 10.1161/CIRCULATIONAHA.120.051171
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E.G-R., G.D-M., J.F.A., E.M.B., M.M.). Centro de Investigación Biomédica en
Red de Enfermedades Cardiovasculares, Spain (J.O., R.R-D., R.R-M., A.M.B.,
J.M.R.). Centro Nacional de Investigaciones Cardiovasculares Carlos III, Madrid,
Spain (M.J.R-R., J.M.R.). Departamento de Farmacología, Universidad Autóno-
ma de Madrid, Instituto de Investigación Hospital La Paz, Spain (R.R-D., C.B-M.,
A.M.B.). Instituto de Investigación Sanitaria-Fundación Jimenez Diaz, Madrid,
Spain (R.R-M. J.L.M-V.). Hospital Universitario Puerta de Hierro, Madrid, Spain.
(R.R-M., J.L.M-V.). Cardiovascular Surgery, Hospital Universitario Marqués de
Valdecilla, IDIVAL, Universidad de Cantabria, Santander, Spain. (J.F.N.). Mas-
sachusetts General Hospital Thoracic Aortic Center, Boston (C.L.L.C., M.E.L.).
Acknowledgments
We thank N.G. Larsson for Tfamfl/fl mice, Dr S. Offermans (Max Planck Institute
for Heart and Lung Research, Bad Nauheim, Germany) for supplying mice ex-
pressing tamoxifen-inducible Cre recombinase specifically in VSMCs (Myh11-
CreERT2), and S. Bartlett for English editing. We also thank the CNIC imaging fa-
cility, A.V. Alonso, and L. Flores for technical support. The genomic data analysis
and statistics were performed by the Genomics and NGS Core Facility at the
Centro de Biología Molecular Severo Ochoa (CBMSO, CSIC-UAM) which is part
of the CEI UAM+CSIC, Madrid, Spain.
J.O. and M.M. designed the research. J.O. performed most of the experi-
ments and analyzed the data. R.R-D., C.B-M. and A.M.B. performed contractility
assays. E.G-R., G.D.M, J.F.A. R.R.M., P.A., E.M.B., and M.J.R-R. provided experi-
mental and technical support. A.F.G., C.E.M.L., J.L.M-V., J.F.N., J.M.R., C.L.L.C.,
and M.E.L. provided reagents. J.O., E.G-R., and M.M. wrote the manuscript.
Sources of Funding
This study was supported by the Fondo de Investigación Sanitaria del Instituto
de Salud Carlos III (PI16/188, PI19/855), the European Regional Development
Fund, and the European Commission through H2020-EU.1.1, European Research
Council grant ERC-2016-StG 715322-EndoMitTalk, and Gobierno de España
SAF2016-80305P. This work was partially supported by Comunidad de Madrid
(S2017/BMD 3867 RENIM-CM) and cofinanced by the European Structural and
Investment Fund. M.M. is supported by the Miguel Servet Program (CP 19/014,
Fundación de Investigación del Hospital 12 de Octubre). J.O., E.G., and R.R-D. are
supported by Juan de la Cierva (FJCI2017-33855, IJC2018-036850-I, and IJCI-
2017-31399, respectively). Support was also provided by Ministerio de Ciencia
e Innovación grants (RTI2018-099246-B-I00 to J.M.R. and PI18/00543 to J.F.N.)
and Comunidad de Madrid and Fondo Social Europeo funds (AORTASANA-CM;
B2017/BMD-3676 to A.M.B., A.F., and J.M.R.). J.M.R. was also funded by Fun-
dacion La Caixa (HR18-00068) and the Marfan Foundation (USA). J.M.R. and
J.L.M.V. were also funded by Centro de Investigación Biomedica en Red Enferme-
dades Cardiovasculares of Ministerio de Ciencia e Innovación (CB16/11/00264).
J.F.N. was funded by Ministerio de Economía y Competitividad (PI18/00543)
and Centro de Investigación Biomedica en Red Enfermedades Cardiovasculares
(CB16/11/00264), and was cofunded by Fondo Europeo de Desarrollo Regional.
Disclosures
None.
Supplemental Materials
Data Supplement Figures I–VI
Data Supplement Table I
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