© 2018. Published by The Company of Biologists Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License

(http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

A comprehensive study of calcific aortic stenosis: from rabbit

to human samples

L. Mourino-Alvarez1, M. Baldan-Martin1, T. Sastre-Oliva1, M. Martin-Lorenzo2, AS.

Maroto2, N. Corbacho-Alonso1, R. Rincon1, T. Martin-Rojas1, LF. Lopez-Almodovar3, G.

Alvarez-Llamas1, F. Vivanco1, LR. Padial4, F. de la Cuesta5, MG Barderas1.

1Department of Vascular Physiopathology, Hospital Nacional de Parapléjicos,

SESCAM, Toledo, Spain.

2Department of Immunology, IIS-Fundacion Jimenez Diaz, Madrid, Spain.

3Cardiac Surgery, Hospital Virgen de la Salud, SESCAM, Toledo, Spain.

4Department of Cardiology, Hospital Virgen de la Salud, SESCAM, Toledo, Spain.

5Centre for Cardiovascular Science.University of Edinburgh.Queen’s Medical Research

Institute.Edinburgh, UK.

Address for correspondence

M.G. Barderas

Laboratorio de Fisiopatología Vascular, Edificio de Investigación, Planta 1, Laboratorio

3, Hospital Nacional de Parapléjicos, SESCAM, 45071 Toledo, España.

e-mail: megonzalezb@sescam.jccm.es FAX: 925247745.

Keywords

Aortic stenosis; cardiovascular; proteomics; rabbit model

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http://dmm.biologists.org/lookup/doi/10.1242/dmm.033423Access the most recent version at First posted online on 10 May 2018 as 10.1242/dmm.033423

Summary statement

Using a rabbit model of calcific aortic stenosis, we have defined a molecular panel of 3

proteins related to osteoblastic differentiation. Additionally, this panel has been

confirmed in human samples.

Abstract

The global incidence of calcific aortic stenosis (CAS) is increasing due, in part, to a

growing elderly population, and poses a great challenge to public health because of the

multiple comorbidities of these patients. Using a rabbit model of CAS, we sought to

characterize protein alterations associated with calcified valve tissue that can be

ultimately measured in plasma as non-invasive biomarkers of CAS.

Aortic valves from healthy and mild stenotic rabbits were analyzed by two-dimensional

difference gel electrophoresis, and selected reaction monitoring was used to directly

measure the differentially expressed proteins in plasma from the same rabbits to

corroborate their potential as diagnostic indicators, and also in plasma from human

subjects, to examine their translatability to the clinical setting.

Eight proteins were found differentially expressed in CAS tissue, but only 3 were also

altered in plasma samples from rabbits and humans: transitional endoplasmic reticulum

ATPase, tropomyosin alpha-1 chain and L-lactate dehydrogenase B chain. Results of

receiver operating characteristic curves showed the discriminative power of the scores,

which increased when the three proteins were analyzed as a panel. Our study shows

that a molecular panel comprising 3 proteins related to osteoblastic differentiation may

have utility as a serum CAS indicator and/or therapeutic target.

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INTRODUCTION

Aortic stenosis is defined as a narrowing of the aortic valve (AV),which results in

reduced blood flow to the body and ultimately in compromised heart function

(Rajamannan et al., 2011). Calcific aortic stenosis (CAS), the most common etiology of

aortic stenosis in Western countries, is characterized by an inflammatory process and

endothelial damage caused by mechanical stress and lipid penetration, leading to

fibrosis and leaflet thickening (Dweck et al., 2012). As the disease progresses, matrix

remodeling and active bone formation occurs, ultimately leading to calcification (Otto,

2006). CAS has a prolonged asymptomatic period defined as aortic sclerosis, during

which time calcification of the valve begins to occur, but with no elevation of the

transvalvular gradient. Nevertheless, once symptoms develop, CAS is rapidly fatal as

there is no effective pharmacologic treatment (Dweck et al., 2012). Patient

management includes balloon valvuloplasty, which only has transient effects, and

aortic valve replacement, either surgical or using transcatheter aortic valve implantation

(TAVI) (Joseph et al., 2016). Accordingly, there is a great unmet need for alternative

therapies to reduce the overall burden of this disease, and efforts have been directed

to controlling CAS progression, as well as to better understand the molecular

mechanisms of CAS to provide potential indicators at early stages of the disease.

CAS is a multifactorial disease, and an important challenge in its study is the presence

of comorbidities, including its increased incidence with age. Animal models have been

instrumental in dissecting the pathogenesis of CAS, as they allow a perfect control of

external factors. In this respect, the rabbit model of aortic stenosis has been particularly

useful because of the similarities between rabbits and humans in terms of valve

histology and lipoprotein metabolism (Cimini et al., 2005, Turk and Laughlin, 2004).

Besides, the existence of osteogenic cells in pathological valves has been previously

described in both humans and rabbits (Kapustin et al., 2011, New and Aikawa, 2013,

Liberman et al., 2008, Drolet et al., 2008).

We have previously applied different strategies to investigate molecular changes taking

place during CAS using diverse biological samples such as plasma (Mourino-Alvarez et

al., 2016a, Gil-Dones et al., 2012), the secretome (Alvarez-Llamas et al., 2013), and

tissue (Mourino-Alvarez et al., 2016b, Martin-Rojas et al., 2016, Martin-Rojas et al.,

2012). In this work, we have directly analyzed AV tissue from mild stenotic rabbits

using a proteomic approach to identify alterations at the molecular level, which may

also be reflected in plasma.

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RESULTS

Development of CAS in the rabbit model

The occurrence of CAS was evaluated by transthoracic echocardiographic examination

(Table S1). As expected, after 12 weeks on a cholesterol-enriched chow plus vitamin

D2 diet, all rabbits from the pathological group (n=7) showed higher peak gradient and

thickened AVs than the control group (n=7, Fig. 1A,B), which confirmed the

development of CAS. By contrast, the interventricular septum and left ventricular free

wall, as well as left ventricular function (reflected by ejection fraction), were not

significantly modified by the diet (Table S1).

Blood analyses were performed at the beginning and during the development of the

study, after 6 and 12 weeks of controlled diet (Table S2 and Fig. 1C). Results showed

that levels of total cholesterol were significantly higher in the pathological group than in

the control group (13.99±0.29 vs 0.53±0.17 g/L, p<0.001). Specifically, the

pathological/control ratio of LDL cholesterol was>58 (13.5±0.55 vs 0.23±0.14g/L,

p<0.001). Significant differences were also found between pathological and control

groups for HDL and non-HDL cholesterol levels at the time of sacrifice (p<0.05).

Histological analysis of AVs from the pathological group revealed the presence of

moderate calcium deposits (as revealed by alizarin red staining),abundant infiltration of

macrophages (RAM11-positive cells; 2.09±1.61% in the pathological group vs

0.015±0.014% in controls, p=0.023), and high expression of α-actin (0.91±0.74% in the

pathological group vs 0.015±0.014% in controls, p=0.018), which is characteristic of

smooth muscle cells and myofibroblasts (Fig. 2). In addition to differences at the

histological level, we also noted that the pathological group had thicker AVs than the

control group, as we have seen in the echocardiographic examination.

Two-dimensional difference gel electrophoresis analysis and differentially

expressed proteins

We used two-dimensional difference gel electrophoresis (2D-DIGE) in combination with

tandem mass spectrometry to compare the relative abundance of proteins extracted

from valve tissue in the two groups. Scanned gel images were analyzed with DeCyder

Differential Analysis Software (GE Healthcare, Chicago, IL, USA), which allows

detection, quantitation, matching, and statistical analysis of the images. Statistical

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analysis (t-test) revealed significant changes in the abundance (p ≤ 0.05 and average

ratio >1.5 or < −1.5) of 15 spots: 5 were up-regulated in CAS tissue and 10 were down-

regulated (Fig. 3). The results were analyzed using Principal Component Analysis

(PCA) to reduce the complexity of the data set and to look for distinctive proteome

profiles in the two study groups. As shown in Fig.S1, pathological and control valves

were separated into two different groups.

Matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometer

(MALDI TOF/TOF) was used to identify the significantly altered spots, which revealed 8

proteins. Calreticulin, transglutaminase-2 and transitional endoplasmic reticulum

ATPase (TERA) were up-regulated in CAS, whereas serum albumin, tropomyosin

alpha-1 chain (TPM-1), L-lactate dehydrogenase B chain (LDHB), myosin light chain 3

and myosin regulatory light chain 2, ventricular/cardiac muscle isoform were down-

regulated (Table 1).

As myosins, TPM-1andLDHB are highly expressed in myocardial tissue (Kamakura et

al., 2013, Lossie et al., 2014, Liu et al., 2016), we confirmed their presence in valve

tissue by immunohistochemistry (Fig.S2).

Selected reaction monitoring

We were able to monitor 5 of these proteins in rabbit plasma by selected reaction

monitoring (SRM) using liquid chromatography tandem-mass spectrometry (LC-

MS/MS). Among them, 3 had a consistent result in the 3 transitions of the 2 peptides

that were measured: TERA, TPM-1 and LDHB. TPM-1 and LDHB were found to be

significantly down-regulated in plasma, whereas TERA was significantly up-regulated

(Table 2 and Fig. 4). We also measured the levels of these proteins in plasma from

patients with AV disease (n=34)and from control subjects (n=12) (characteristics of the

patient study groups are shown in Table 3). This analysis showed that TERA, TPM-1

and LDHB were also significantly altered in human subjects and followed the same

trend as the rabbit plasma (Table 2 and Fig. 4).

Results from rabbit and human plasma were used to assess the sensitivity and

specificity of these potential markers by individual receiver operating characteristic

(ROC) curves. In rabbits, the area under the curve (AUC) was 1.0 with significant

values (p<0.01) for all peptides. In human plasma, the AUC was higher than 0.73 and

the p-value below 0.037 in all cases. Moreover, when the 3 proteins were combined,

the AUC increased to1.0 and the p-value decreased to6.28×10-6 (Fig. 5).

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DISCUSSION

Animal models are of great utility for studying several diseases as they allow the

control of the differences between the experimental groups. This is especially important

in the setting of CAS given the etiology of the disease; as it appears at advance ages, it

is usually accompanied by other comorbidities such as hypertension or diabetes. Our

rabbit model was based on a hypercholesterolemic diet supplemented with vitamin D,

which has been shown to be effective for the study of the evolution of the early phases

of AV disease (Drolet et al., 2003) and also to investigate the potential prevention of

progression to severe stages (Busseuil et al., 2008, Drolet et al., 2003). The increase in

the transvalvular gradient confirmed the development of CAS in the pathological group,

which was concomitant with a significant increase in cholesterol (total, LDL, HDL and

non-HDL), characteristics that have been previously described in patients with CAS

(Kamath and Rai, 2008, Akat et al., 2010). By contrast, the echocardiography study

showed no development of hypertrophy or impaired systolic function of the left

ventricle, which is consistent with development of mild CAS rather than severe disease

(Seiler and Jenni, 1996, Mihaljevic et al., 2008). Importantly, the animal model allowed

us to study valve tissue against a background of mild calcification levels, as it is difficult

to obtain mild calcified valves from patients since AV replacement is recommended

only at advances stages of CAS, when symptoms appear due to severe damage in

valve mobility.

Our protein analysis revealed 8 proteins of interest, of which 3 were significantly altered

as shown by SRM analysis of plasma both from rabbits and patients: TPM-1 and LDHB

followed the same trend in plasma and tissue, whereas TERA was up-regulated in

tissue and down-regulated in both rabbit and human plasma.

Structural proteins such as TPM-1 are part of the cardiac muscle and the contractile

cytoskeleton of various cell types including fibroblasts and endothelial cells, and is

essential for the maintenance of the endothelial barrier (Mehta and Malik, 2006).

Overexpression of TPM-1 has been shown to stabilize the structure of actin filaments,

helping to preserve endothelial barrier function under oxidative stress conditions

(Gagat et al., 2014). Moreover a reduction in its expression has been previously related

to a decrease in the contraction of actin filaments in arteries with arteriosclerosis

(Wang et al., 2011). The reduction of TPM-1 levels may also relate to the loss of AV

flexibility as occurs in arteriosclerosis, being indicative of the differentiation of the

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valvular interstitial cells to osteoblasts, which have no contractile ability (Lei et al.,

2014, Park et al., 2009).

Valve calcification is also related to higher endoplasmic reticulum stress (ERS) (Cai et

al., 2013). The authors of this study showed that oxidized LDL promotes osteoblastic

differentiation through the activation of the ERS pathway. In the present study, we

found higher levels of TERA, also named valosin-containing protein, in the AV tissue of

the pathological group. TERA is responsible for exporting misfolded proteins to the

endoplasmic reticulum, where they accumulate and trigger ERS (Jarosch et al., 2002).

In a similar way to TPM-1, the increase of TERA found in our study points to the

differentiation of valvular interstitial cell to osteoblasts, which has been previously

related to ERS (Cai et al., 2013).

In valve tissue, oxygen requirements exceed the amount deliverable by diffusion from

the cusp surfaces alone, and so vasculature is needed to maintain a sufficient oxygen

supply to the cells (Weind et al., 2000, Weind et al., 2002). It is therefore reasonable to

assume that tissue thickening may lead to a reduction in the amount of oxygen

received by these cells. In hypoxic conditions, lactate is formed from pyruvate via

LDHA. LDHB is a heart-specific isoform that catalyzes the same reaction in aerobic

conditions (Buono and Lang, 1999).This isoform has been shown to be downregulated

in conditions of oxygen deprivation(Rossignol et al., 2003, Kay et al., 2007), which is in

accordance with our results in CAS tissue and in serum from rabbits and humans with

CAS.

We believe that the alterations we have found at the tissue level are significant as they

provide molecular information about the mechanisms that take place within the valve.

Moreover, these proteins may serve as potential therapeutic targets to slow down the

progression of the disease, although more functional analysis is needed. Of particular

interest is the opposite trend we found for expression of TERA in tissue (higher in

stenotic valves) and plasma (lower levels in CAS rabbits/subjects). TERA is commonly

found in extracellular vesicles in several types of cells, including endothelial cells

(Peterson et al., 2008) or lymphocytes (Miguet et al., 2006). It has also been identified

in microparticles derived from human atherosclerotic plaques (Mayr et al., 2009). Given

the increasing importance of extracellular vesicles in cardiovascular disease (Yin et al.,

2015, Chen et al., 2018, Badimon et al., 2017), these variations in TERA levels should

be further studied as they could be indicative of differential tissue and/or cell vesicle

release during the development of CAS.

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We have demonstrated the great potential of SRM to quantify differences in proteins

across multiple samples. In rabbit samples, peptides from TPM-1 and LDHB do not

have the same ratio estimations, probably because 2 peptides are outside the linear

range of the assay (Bao et al. Methods, 2013). Special careful was taken to avoid this

effect in plasma samples, something crucial for the evaluation of the potential utility as

diagnostic markers of these proteins. As shown in the ROC curves, each of the verified

proteins has sufficient sensitivity and specificity to discriminate between control and

pathological subjects (AUC>0.73), pointing to their potential utility as diagnostic

markers. It should be noted that the combined measurement of the three proteins as a

panel has greater discriminate power (AUC=1.0), presenting a very high capacity to

assign subjects to their corresponding study groups. Analysis in plasma samples may

facilitate the translation of this panel to the clinical field, as blood samples are easy to

obtain and are minimally invasive compared with biopsies and surgical procedures. Our

use of LC-MS/MS for verification has not been by chance. The use of this technology in

routine clinical laboratories has witnessed unprecedented growth during the last two

decades due not only to its high specificity, sensitivity and high-throughput potential,

but also because it is faster and more flexible than classical immunoassays (Grebe and

Singh, 2011, Leung and Fong, 2014). Therefore, it is foreseeable that LC-MS/MS will

become a powerful tool in routine clinical laboratories.

Some limitations of this work should be highlighted. One of the most important

challenges of using animal models is the notable species differences between animal

models and humans. Nevertheless, CAS in vitamin D2-supplementation models is

histologically and hemodynamically similar to the human disease, involving

fibrosis/calcification, inflammatory response and endothelial dysfunction (Ngo et al.,

2008) as well as changes in cardiac function, as shown here. Also, the rabbit model

has a greater translational strength than murine models. Clearly, the analysis of human

samples has the disadvantage that the underlying pathological and physiological

conditions cannot be controlled, especially when studying elderly patients as they

present more concomitant diseases. Finally, according to the classical development

biomarker pipeline (Surinova et al., 201), these proteins should be validated in an

independent cohort of at least 100 subjects prior to clinical evaluation.

In sum, we have defined a new molecular panel that can be measured in plasma using

an extremely reproducible and reliable method, such as SRM, indicating its potential for

implementation in the clinic. Nevertheless, it will be necessary to perform further

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studies to assess some remaining aspects. It will be important to use in vitro and in

vivo models to clearly define the role of these proteins in tissue. If, as we hypothesize,

3 of them are related to osteoblastic differentiation, they may be interesting as putative

therapeutic targets to reduce calcium deposition. Besides, studies about the implication

of ERS in valve calcification are scarce so it would be interesting to deepen these

mechanisms and to study the specific role of TERA in AV calcification. Finally, a

prospective study in a larger cohort of subjects with different degrees of AV damage,

from mild sclerosis to severe stenosis, should be carried out to validate the diagnostic

and prognostic value of these indicators. If these candidates are suitable for clinical

evaluation, it may lead to a considerable improvement in patient management,

reducing the burden of CAS in society.

MATERIALS AND METHODS

Animal model

Male New Zealand white rabbits (Oryctolagus cuniculus) weighing 2–2.5 kg were

divided into two groups: animals in the control group (n=7) were fed with normal rabbit

chow; animals in the pathological group (n=7) were fed with 1% cholesterol-enriched

chow plus 50,000 IU/kg vitamin D2 (Harlan, Indianapolis, IN, USA). All animals were

fed ad libitum for 12 weeks (Drolet et al., 2008). Echocardiographic evaluations of the

AV were performed at t=0, t=6 weeks and t=12 weeks, to ensure the establishment of

CAS. Blood was drawn into EDTA tubes through the marginal vein of the ear at the

same time for the measurement of cholesterol and triglycerides. After the 12-week

period, animals were sedated with an injection of ketamine (100 mg/kg) and xylazine

(20mg/kg) and then euthanized by injection of pentobarbital (50 mg/kg) directly into the

heart. AVs were immediately harvested, rinsed in saline buffer and processed for

analyses. When the analysis was not performed immediately, tissues were stored at -

80°C.

The study was conducted in accordance with the Principles of Laboratory Animal Care

and all experimental procedures were approved by the Animal Care and Use

Committee of the IIS-Fundación Jiménez Díaz, according to the guidelines for ethical

care of the European Community.

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Echocardiography

Ultrasound video images were obtained using the HD11XE echocardiographic system

(Philips Medical Imaging, Best, the Netherlands) and a neonatal S12-4 ultrasound

imaging probe, with an extended frequency range of 4 to 12 MHz. A parasternal long-

axis view was used to measure valvular thickness and left ventricle parameters. Left

ventricular ejection fraction and fractional shortening were calculated from

measurements of the left ventricular internal diameter in systole and diastole.

Additionally, aortic outflow velocity was registered using continuous-wave Doppler

echocardiograpy from apical planes, and the peak gradient was calculated using the

Bernoulli equation (Baumgartner et al., 2009).

Tissue staining

One leaflet of each valve was fixed in 4% buffered formalin for 24 hours and then

embedded in paraffin. Paraffin-embedded sections were subjected to hematoxylin-

eosin and alizarin red staining for visualization of calcium deposits. The following

monoclonal antibodies were used for immunohistochemistry: RAM11 for macrophages

(dilution 1:100, M0633, DAKO, Santa Clara, CA, USA), α-actin for vascular smooth

muscle cells (1:100, M0851, DAKO), tropomyosin α (1:25,sc-376541, Santa Cruz

Biotechnology, Dallas, TX, USA), L-lactate dehydrogenase B chain (1:200, sc-100775,

Santa CruzBiotechnology), myosin light chain 3 (1:2000, ab680, Abcam, Cambridge,

UK), and myosin regulatory light chain 2 (1:200, ab89594, Abcam). In control

experiments, no primary antibody was added. Non-specific binding was prevented by

incubation with normal goat serum (for RAM11) or 10% bovine serum albumin (for the

remainder) for 1 h, and non-specific peroxidase activity was blocked with 3% hydrogen

peroxidase for 5 min. Incubation with primary antibodies was performed for 1 h at room

temperature. The slides were then incubated with horseradish peroxidase-conjugated

polyclonal goat anti-mouse antibodies (dilution 1:100, P0447,Dako) for 30 min, and the

chromogenic reaction was developed using 3,3′diaminobenzidine (DAB). Sections were

then counterstained with hematoxylin prior to dehydration and coverslipping. For an

impartial analysis of the DAB staining, an orthonormal transformation of the RGB

images using an ImageJ plugin (NIH) based on Ruifrok and Johnston’s method for

color deconvolution was performed (Ruifrok and Johnston, 2001).

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Proteomic analysis using two-dimensional difference gel electrophoresis

One AV leaflet was ground into powder in liquid N2 with a mortar. Proteins were then

extracted using 7M urea, 2M thiourea, 4% CHAPS and 1% dithiothreitol (DTT) and the

homogenate was centrifuged to precipitate tissue debris. The supernatant was

collected and the protein concentration was determined using the Bradford assay.

Before proteomic analysis, the required amount of protein was subjected to a cleaning

step by precipitation using the 2-D Clean-Up kit (GE Healthcare, Chicago, IL, USA) and

resuspended in rehydratation buffer (7M urea, 2M thiourea, 4% CHAPS, 30 mM Tris) to

a final concentration of 7 mg/ml. Proteins were then labeled according to the

manufacturer’s instructions (GE Healthcare). Briefly, 50 µg of protein from each AV

extract was labeled with 400 pmol of N-hydroxysuccinimide esters of Cy3 or Cy5

fluorescent cyanine dye for 30 min on ice in the dark. An internal standard containing

equal amounts of all experimental samples was labeled with 400 pmol of N-

hydroxysuccinimide Cy2 dye. Reactions were then quenched with 0.2 mM lysine.

Labeled protein extracts were combined according to the experimental design (Table

S3), diluted in rehydration buffer (30 mM Tris, 7M urea, 2M thiourea, 4% CHAPS) with

2% DTT and 1% ampholytes (IPGbuffer 4–7, GE Healthcare) and applied to 24-cm pH

4–7 IPG strips. After passive rehydration, the first dimension was run on the IPGphor

IEF II System (GE Healthcare), as follows: 500 V during 1 h, a linear gradient to 1000 V

over 2 h, a linear gradient to 8000 V over 3 h, and 8000 V until 96,000 V/h. After the

first dimension, the strips were equilibrated in SDS-equilibration buffer (1.5M Tris-HCl

pH 8.8, 6M urea, 87% glycerol and 2% SDS) using a two-step protocol for reduction

(by adding 1% DTT) and alkylation (by adding 2.5% iodoacetamide) of thiol groups.

Proteins were then separated on 10% acrylamide/bisacrylamide gels using an

EttanDalt Six device (GE Healthcare) (Laemmli, 1970).

Image acquisition and analysis

Gels were scanned on a Typhoon 9400 fluorescence gel scanner (GE Healthcare)

using appropriate individual excitation and emission wavelengths, filters and

photomultiplier values sensitive for each of the Cy3, Cy5 and Cy2 dyes.

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Images were analyzed using DeCyder software v6.5 (GE Healthcare). The Differential

In-gel Analysis module co-detected the 3 images of each gel (spot maps from the

internal standard and the two samples), measured the spot abundance in each image

and expressed these values as Cy3/Cy2 and Cy5/Cy2 ratios. The Biological Variation

Analysis module enabled the matching of these spot maps, the comparison of the

Cy3/Cy2 and Cy5/Cy2 ratios and the statistical analysis, to determine changes in

expression levels. Only protein spots with >1.5-fold difference in abundance and with

p-values below 0.05 (Student´s t-test) were considered as proteins of interest. Finally, a

multivariate analysis was performed by PCA using the Extended Data Analysis module.

A pattern analysis hierarchical classification was also obtained using the Pearson

coefficient based on the spots present in 90% of all the gels.

In-gel digestion and protein identification by matrix-assisted laser

desorption/ionization time-of-flight mass spectrometry

Differentially expressed protein spots were excised manually from the 2D-DIGE gels,

which were previously stained with Oriole TM fluorescent gel stain (Bio-Rad, Hercules,

CA, USA). Additionally, a preparative gel using 400 µg of total protein was also

prepared for identification of small or low abundant proteins, using the same

electrophoretic parameters. Spots were automatically digested with the Ettan Digester

workstation (GE Healthcare) and identified at the Proteomic Unit of Hospital Nacional

de Parapléjicos. The digestion was performed according to Shevchenko et al.

(Shevchenko et al., 1996)with minor modifications and, after digestion at 37ºC

overnight, the peptides were extracted with 60% acetonitrile (ACN) in 0.1% formic acid.

Samples were dried in a speedvac and resuspended in 98% water with 0.1% formic

acid and 2% ACN. An aliquot of each digestion was mixed with an aliquot of the matrix

solution (3 mg/mL matrix α-cyano-4-hydroxycinnamic acid in 30% ACN, 15% 2-

propanol and 0.1% trifluoroacetic acid), and this was pipetted directly on a 384 Opti-

TOF 123 × 81mm stainless steel sample plate and analyzed on a 4800 Plus MALDI

TOF/TOF mass spectrometer (Applied Biosystems, Foster City, CA, USA).

MALDI-MS/MS analysis and database searching

MALDI-MS/MS data were obtained using an automated analysis loop in the MALDI

TOF/TOF Analyzer. MALDI-MS and MS/MS data were combined using GPS Explorer

Software Version 3.6 to search a non-redundant protein database (Swissprot 56.5) with

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Mascot software version 2.2 (Matrix Science, London, UK)(Perkins et al., 1999)

applying the following settings: 50 ppm precursor tolerance, 0.6 Da MS/MS fragment

tolerance, 1 missed cleavage allowed, carbamidomethyl cysteines and methionine

oxidation as modifications. The MALDI-MS/MS spectra and database search results

were manually inspected in detail using the aforementioned software.

Patient selection and blood extraction

Peripheral blood samples were collected from control subjects (n=12) and patients with

severe CAS (n=34) obtained from subjects who underwent scheduled AV replacement

atHospital Virgen de la Salud (Toledo, Spain). In patients, AV area (0.74±0.19 cm2),

ejection fraction (57.13±9.83%) and mean gradient (48.16±18.06 mmHg) were

assessed using transthoracic echocardiography. Blood samples were always taken

prior to surgery. Subjects were selected to avoid significant differences between the

groups in terms of the following main cardiovascular risk factors: age, gender, obesity,

hypertension, dyslipidemia and diabetes (Table 3). Samples from patients with bicuspid

AV, concomitant aortic stenosis and aortic regurgitation or mitral valve disease were

excluded.

The patient study was carried out in accordance with the recommendations of the

Helsinki Declaration and was approved by the ethics committee at the Hospital Virgen

de la Salud. Signed informed consent was obtained from all subjects. Blood samples

(28 ml) were drawn into EDTA-containing tubes and centrifuged at 1125 × g for 15 min

and the resulting supernatant was immediately frozen at -80°C until analysis.

Selected reaction monitoring

Proteins from plasma samples were reduced and alkylated by incubating with 100 mM

DTT and 550 mM iodoacetamide in 50 mM ammonium bicarbonate, respectively.

Proteins were digested in 50 mM ammonium bicarbonate, 15% acetonitrile with

sequencing grade modified porcine trypsin, at a final concentration of 1:50

(trypsin:protein). After overnight digestion at 37ºC, 2% formic acid was added and

samples were cleaned with Pep‐Clean spin columns (Pierce, Waltham, MA, USA).

Tryptic digests were dried in a speedvac and resuspended in 2% ACN, 2% formic acid

prior to MS analysis.

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The LC/MS/MS system consisted of a TEMPO nano LC system (Applied Biosystems)

combined with a nano LC Autosampler and coupled to a modified triple quadrupole MS

system (Applied Biosystems 4000 QTRAP LC/MS/MS). Three replicate injections (4μl

containing 1μg of protein) were made for each sample using mobile phase A (2%

ACN/98% water, 0.1% formic acid [FA]) with a flow rate of 10μL/min for 5min. Peptides

were loaded onto a μ‐Precolumn Cartridge (Acclaim Pep Map 100 C18, 5 μm, 100Å;

300 μm i.d.× 5mm, LC Packings, Amsterdam, the Netherlands) to preconcentrate and

desalt samples. Reverse-phase liquid chromatography was achieved on a C18 column

(Onyx Monolithic C18, 150 × 0.1mm i.d., Phenomenex, Torrance, CA, USA) in a

gradient of phase A and phase B (98% ACN/2% water, 0.1% FA). Peptides were eluted

at a flow rate of 900 nL/min in the following steps: 5–45% B for 60 min, 45–95% B for 1

min and finally 95% B for 4 min. The column was then regenerated with 5%B for 15

min. Both the TEMPO nano LC and 4000 QTRAP systems were controlled by Analyst

Software v.1.5.2. The mass spectrometer was set to operate in positive ion mode with

ion spray voltage of 2800V and a nanoflow interface heater temperature of 80ºC.

Source gas 1 and curtain gas were both set to 20, and nitrogen was applied as both

curtain and collision gases. For peptide selection, theoretical SRM transitions were

designed using Skyline software v3.1.0.7382 (MacCoss Lab, Seattle, WA, USA) and

Unique Peptide function from that software was used to verify that theoretical tryptic

peptide sequences were proteotypic. A sample containing a mixture of all the proteins

of interest was digested and analyzed with a MIDAS acquisition method, which

combines an MRM scan with a full MS/MS product ion scan to allow examination of all

fragment ions in the same spectrum. Peptides with six co-eluting transitions with a

signal‐to‐noise ratio over 5 were considered for the analysis. Among these, the three

most intense transitions for each peptide were selected for the quantification,

optimizing collision energy and dwell times to obtain maximum transmission efficiency

and sensitivity for each one (Table S4). Skyline software was also used to calculate the

peptide abundance on the basis of peak areas after integration.

Statistical analysis

Statistical analyses were performed using SPSS 15.0 for windows software (SPSS

Inc., Chicago, IL, USA). Continuous variables, such as age, are expressed as mean ±

standard deviation. After demonstrating normal distribution of the population using a

Kolmogorov-Smirnov test, comparison of means was performed using Student's t-test.

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Differences in variables in rabbits before and after the diet were analyzed using paired

t-test. Discrete variables, such as sex or the presence/absence of risk factors, are

expressed as percentages. In these cases, Fisher's exact test was used for

comparison of the groups. ROC curves were generated using SPSS 15.0.

For SRM, only peptides with significant p-values in at least two of the three measured

transitions were considered significant. Results from rabbit plasma are shown as t=12

weeks/t=0 ratio.

ACKNOWLEDGMENTS

We thank the Proteomics Unit Hospital Nacional de Parapléjicos for assistance with the

protein identification and the Microscopy Unit Hospital Nacional de Parapléjicos for

assistance with the image analysis.

COMPETING INTERESTS

We do not have conflicts of interest.

FUNDING INFORMATION

This work was supported by grants from the Instituto de Salud Carlos III [PI11-02239,

PI14-01917, grant PRB3 (IPT17/0019-ISCIII-SGEFI/ERDF)], FONDOS FEDER

[RD06/0014/1015, RD12/0042/0071] and the Spanish Society of Cardiology. These

results are lined up with the Spanish initiative on the Human Proteome Project

(SpHPP).

AUTHOR CONTRIBUTION STATEMENT

L. Mourino-Alvarez and M. Baldan-Martin performed 2D-DIGE and SRM experiments,

including statistical analyses; T. Sastre-Oliva and N. Corbacho-Alonso performed the

immunohistochemistry experiments; M. Martin-Lorenzo, AS. Maroto, G. Alvarez-

Llamas and F. Vivanco collaborated in the development of the animal model and

helped with rabbit sample preparation; R. Rincon and T. Martin-Rojas collaborated with

validation experiments and data analysis. All the work related to the selection, study

and follow-up of patients was carried out by Lopez-Almodovar and LR. Padial; they

also helped with echocardiography. Finally, F. de la Cuesta and MG. Barderas

designed and supervised the entire study.

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TABLES

Table 1. List of proteins identified by mass spectrometry (MALDI TOF/TOF) found

significantly differentin 2D-DIGE analysis. The table shows spot number, accession

number according to Uniprot, protein name, statistical results (ratio CAS/C and p-value

according to Student's t-test), main function, score obtained in the identification using

MASCOT, sequence coverage (%) and number of matched peptides.

Accession

number Name CAS/C

p-

value Function MASCOT

Seq.

Coverage

Matched

peptides

1 P15253 Calreticulin 2.38 0.013 Chaperone 260 82.2 26

2 G1SK42

Protein-glutamine

gamma-

glutamyltransferase 2

1.85 0.025 Acyltransferase 464 45.4 33

3 G1SR03

Transitional

endoplasmic reticulum

ATPase

1.67 0.038 Hydrolase 175 42.4 26

4 P49065 Serum albumin -1.74 0.011 Antioxidant/

Transport 318 67.1 33

8 P58772 Tropomyosin alpha-1

chain -5.28 0.046

Muscular

protein 85 34.5 9

10 P13490 L-lactate

dehydrogenase B chain -1.67 0.023 Oxidoreductase 94 51.2 13

14 G1T375 Myosin light chain 3 -5.41 0.040 Muscular

protein 196 68.4 14

15 Q7M2V4

Myosin regulatory light

chain 2,

ventricular/cardiac

muscle isoform

-6.03 0.041 Muscular

protein 93 53.9 11

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• A

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uscr

ipt

Table 2. Results from plasma analyses using SRM. Shown are the peptides and

transitions measured for each protein and the statistical analyses for each transition,

including mean and p-value.

Species Protein Peptide sequence Fragment

ion

Control

mean±SEM CAS mean±SEM

p-

value

Rabbit

TPM-1

LVIIESDLER

y5 1.27±0.17 0.57±0.13 0.003

y6 1.27±0.17 0.60±0.10 0.005

y9 1.28±0.21 0.64±0.11 0.012

SIDDLEDELYAQK

y5 1.01±0.04 0.11±0.03 <0.001

y6 1.05±0.07 0.13±0.03 <0.001

y8 1.05±0.08 0.13±0.03 <0.001

TERA

GDDLSTAILK

y3 1.38±0.15 0.74±0.08 0.003

y4 1.27±0.15 0.61±0.06 0.002

y6 1.17±0.10 0.69±0.08 0.003

MDELQLFR

y2 1.33±0.16 0.64±0.07 0.002

y3 1.27±0.14 0.68±0.11 0.005

y5 1.39±0.17 0.69±0.11 0.004

LDHB

FIIPQIVK

y3 1.06±0.18 0.58±0.10 0.023

y5 1.00±0.11 0.59±0.12 0.020

y6 1.08±0.15 0.68±0.13 0.039

MVVESAYEVIK

y7 4.45±1.43 0.84±0.13 0.032

y8 6.16±3.65 0.65±0.08 0.102

y9 4.59±1.61 0.94±0.21 0.043

Human

TPM-1 QLEDELVSLQK

y8 8.58E-04±1.26E-04 4.73E-04±4.22E-05 0.006

y6 4.09E-04±8.90E-05 3.00E-04±3.12E-05 0.149

y5 5.05E-04±7.84E-05 3.06E-04±3.24E-05 0.007

TERA

LEILQIHTK

y6 4.37E-03±6.52E-04 2.66E-03±1.95E-04 0.013

b5 1.04E-02±1.52E-03 6.37E-03±4.51E-04 0.013

b6 5.07E-03±7.32E-04 3.12E-03±2.24E04 0.012

GGNIGDGGGAAD

R

y7 1.74E-02±3.66E-03 9.12E-03±8.08E-04 0.024

b9 1.06E-03±2.16E-04 5.58E-04±5.12E-05 0.021

b12 4.60E-03±9.39E-04 2.46E-03±2.27E-04 0.023

LDHB

MVVESAYEVIK

y9 3.82E-02±6.33E-03 2.24E-02±1.60E-03 0.016

y8 1.18E-02±1.96E-03 6.91E-03±4.76E-03 0.016

b8 5.08E-02±7.97E-03 3.00E-02±2.04E-03 0.013

GLTSVINQK

b6 2.64E-02±4.20E-03 1.69E-02±1.22E-03 0.024

b7 1.30E-01±2.05E-02 8.32E-02±6.06E-03 0.024

b8 9.18E-03±1.41E-03 5.95E-03±4.30E-04 0.023

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Table 3. Clinical characteristics of the subjects used in the validation phase with

human samples. M/F, male/female; AHT, arterial hypertension.

Control (n=12) CAS patients (n=34) p-value

Age 65.0±21.96 74.9±8.10 0.47

Gender (%M/F) 58/42 50/50 0.74

% Obesity 8 26 0.25

% AHT 58 82 0.12

% Dyslipidemia 25 59 0.09

% Diabetes 17 32 0.46

% Smoker 0 21 0.17

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Figures

Figure 1. Representative echocardiograms from controls (A) and pathological (B)

rabbits after 12 weeks of diet. Doppler velocity is shown in the upper images (blue

arrows), whereas the aortic valves are shown by white arrows in the lower images. It

can be observed that pathological rabbits have higher Doppler velocity (and,

subsequently, higher transvalvular gradient) and thicker aortic valves. Aorta, left

ventricle (LV) and right atrium (RA) are indicated in the figure. Results from blood

analyses are also shown (C) and significant differences are marked: p<0.05 (*) or

p<0.001 (***). P-values were calculated by comparing each corresponding time point to

t=0 using a paired t-test. Red lines: pathological group; green lines: control group.

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Figure 2. Histology of the aortic valves from pathological and control rabbits. Note the

increased valve thickness (hematoxylin and eosin staining [H&E]; A and B) in

pathological rabbits, and calcium deposits by alizarin red staining (arrow, C and D).

Macrophages (RAM11; E-G) and SMC and/or myofibroblasts (α-actin; H-J) staining is

more intense in the pathological group (arrows). Scale bar corresponds to 200µm in

100× images and 50µm in 400× images. * Aortic valve.

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Figure 3. Master gel of rabbit valve 2D-DIGE showing 15 differential protein spots

between control and pathological groups .Spots that are increased in the pathological

group are shown in red whereas spots that are decreased in this group are shown in

green.

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Figure 4. Plasma analysis using SRM in rabbit (A) and human (B) samples. SRM

analyses allowed the measurement of tropomyosin-1 (TMP-1), transitional endoplasmic

reticulum ATPase (TERA) and L-lactate dehydrogenase B chain (LDHB). All the

transitions were used to calculate mean intensity of each peptide. Relative abundance

is shown (100% corresponds to control group).

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Figure 5. ROC curves of tropomyosin-1 (TMP-1), transitional endoplasmic reticulum

ATPase (TERA) and L-lactate dehydrogenase B chain (LDHB) are shown in the upper

part of the figure. ROC curve of the combined proteins is shown in the bottom image.

When these proteins are combined, obtained panel is more sensitive and specific, so it

would be more useful that individual proteins for the development of clinical diagnostic

tools. In all cases, the transition of the most significant peptide is represented. Area

under the curve (AUC) and p-value are shown.

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Table SM.1.Values for echocardiographic measurements in rabbits at different times of the model (start point, 6 and 12 weeks).Ejection fraction and fractional shortening has been calculated from measurements for the left ventricular internal diameter while peak gradient has been calculated from maximal aortic outflow velocity. Changes were analyzed using paired t-test. Ao, aortic diameter; AV, thickness of the aortic valve; CAS, calcific aortic stenosis;EF, ejection fraction; FS, fractional shortening; IVSd and IVSs, thickness of the interventricular septum in diastole and systole, respectively; LVFWd and LVFWs, thickness of the left ventricular free wall in diastole and systole, respectively; PG, peak gradient.

Group Mean±Standard deviation Reference

interval1

p-values

t=0 t=6 t=12 t=0 vs t=12 t=0 vs t=6 t=6 vs t=12

AV (mm) Control rabbits 0.39±0.05 0.38±0.03 0.40±0.05

- 0.98 0.63 0.42

CAS rabbits 0.37±0.05 0.52±0.07 0.54±0.07 0.02 0.05 0.41

IVSd (mm) Control rabbits 2.41±0.35 2.86±0.82 2.48±0.54

1.74-3.74 0.97 0.29 0.66

CAS rabbits 2.22±0.18 2.68±0.81 2.93±0.68 0.06 0.11 0.91

IVSs (mm) Control rabbits 4.41±0.55 4.29±0.78 3.95±0.31

2.64-5.38 0.13 0.76 0.45

CAS rabbits 4.21±0.39 4.77±0.76 3.93±0.62 0.62 0.40 0.03

LVFWd (mm)

Control rabbits 3.54±3.40 2.98±0.89 2.55±0.36 1.72-3.84

0.46 0.69 0.47

CAS rabbits 2.25±0.38 3.15±0.83 3.14±0.91 0.12 0.09 0.94

LVFWs (mm)

Control rabbits 3.96±0.32 4.11±0.63 3.77±0.35 2.54-4.58

0.36 0.59 0.43

CAS rabbits 3.98±0.29 4.23±0.27 3.91±0.93 0.91 0.25 0.43

Ao (mm) Control rabbits 6.85±0.34 6.89±0.76 6.92±0.73

6.39-9.41 0.90 0.90 0.77

CAS rabbits 6.63±0.28 7.38±0.57 6.80±0.57 0.92 0.20 0.12

EF (%) Control rabbits 63.75±7.00 62.62±5.65 62.47±4.83

58.73-83.51 0.91 0.72 0.74

CAS rabbits 65.81±7.73 62.19±5.05 50.12±11.48 0.19 0.58 0.07

FS (%) Control rabbits 31.79±4.81 31.31±4.07 30.77±3.34 27.39-46.95 0.88 0.83 0.60

1GIANNICO AT ET.AL. DETERMINATION OF NORMAL ECHOCARDIOGRAPHIC, ELECTROCARDIOGRAPHIC, AND RADIOGRAPHIC CARDIAC PARAMETERS IN THE CONSCIOUS NEW

ZEALAND WHITE RABBIT.JOURNAL OF EXOTIC PET MEDICINE 24 (2015), PP 223–234

Disease Models & Mechanisms 11: doi:10.1242/dmm.033423: Supplementary information

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CAS rabbits 33.40±6.12 30.89±3.25 23.47±6.74 0.20 0.59 0.07

PG (mmHg) Control rabbits 1.57±0.75 1.72±0.32 1.34±0.59

0.89-4.28 0.24 0.58 0.07

CAS rabbits 0.95±0.86 1.96±0.49 2.44±1.23 0.00 0.04 0.34

Disease Models & Mechanisms 11: doi:10.1242/dmm.033423: Supplementary information

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Table SM.2. Blood analysis results from rabbit model samples. Different values at the start of the model, 6 and 12 weeks are shown.Paired t-test was used to compare samples from t=0 and t=12 and corresponding p-values are shown.

Control group Pathological group

t=0 t=6 t=12 p-value t=0 t=6 t=12 p-value

Cholesterol (g/L) 0.63±0.12 0.49±0.15 0.53±0.17 0.30 0.72±0.19 14.14±0.70 13.99±0.29 1.9x10-09

Triglyceride (g/L) 0.91±0.30 0.59±0.15 0.64±0.23 0.06 13.86±0.63 17.04±0.68 16.57±0.99 0.54

HDL (g/L) 0.30±0.07 0.26±0.10 0.22±0.07 0.05 0.28±0.08 0.20±0.07 0.13±0.12 0.03

LDL (g/L) 0.15±0.09 0.12±0.07 0.23±0.14 0.28 0.16±0.14 13.66±0.67 13.53±0.55 2.0x10-08

Non-HDL (g/L) 0.33±0.07 0.23±0.07 0.36±0.12 0.67 0.43±0.16 13.95±0.67 13.86±0.39 3.1x10-09

Disease Models & Mechanisms 11: doi:10.1242/dmm.033423: Supplementary information

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Table SM.3.Experimental design for 2D-DIGE analysis. All gels included the internal standard (IS) and two additional tissue samples from the pathological and control group. PR=Pathological rabbit; CR=Control rabbit.

Gel number Cy2 (IS) Cy3 Cy5

1 Pool PR1 CR1

2 Pool CR2 PR2

3 Pool PR3 CR3

4 Pool CR4 PR4

5 Pool PR5 CR5

6 Pool CR6 PR6

Disease Models & Mechanisms 11: doi:10.1242/dmm.033423: Supplementary information

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Table SM.4. List of protein monitored by SRM including the experimental parameters.

Specie Protein Name

Accession Peptide sequence Precursor

m/z Product

m/z Collision energy

Fragment ion

Rabbit

TPM-1 P58772

LVIIESDLER 593.84

619.30 31.1 y5

748.35 31.0 y6

1073.58 31.1 y9

SIDDLEDELYAQK 769.86

622.36 38.9 y5

751.40 38.9 y6

995.47 38.9 y8

TERA G1SR03

GDDLSTAILK 516.78

373.28 27.7 y3

444.32 27.7 y4

632.40 28.0 y6

MDELQLFR 526.27

322.19 28.2 y2

435.27 28.2 y3

676.41 28.0 y5

LDHB G1TYA7

FIIPQIVK 479.31

359.27 26.1 y3

584.38 26.0 y5

697.46 26.0 y6

MVVESAYEVIK 634.33

809.44 33.0 y7

938.48 32.9 y8

1037.55 32.9 y9

Human TPM-1 P09493 QLEDELVSLQK 651.35 934.51 32.9 y8

687.44 32.9 y6

Disease Models & Mechanisms 11: doi:10.1242/dmm.033423: Supplementary information

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Specie Protein Name

Accession Peptide sequence Precursor

m/z Product

m/z Collision energy

Fragment ion

574.36 32.9 y5

TERA P55072

LEILQIHTK 547.83

739.45 27.0 y6

597.36 27.0 b5

710.44 27.0 b6

GGNIGDGGGAADR 558.76

603.28 27.6 y7

685.29 27.6 b9

942.39 27.6 b12

LDHB P07195

MVVESAYEVIK 634.33

1037.55 31.9 y9

938.48 31.9 y8

909.40 31.9 b8

GLTSVINQK 480.28

571.34 23.1 b6

685.39 23.1 b7

813.45 23.1 b8

Disease Models & Mechanisms 11: doi:10.1242/dmm.033423: Supplementary information

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Figure SM.1. Score plot obtained in the principal

Figure SM.1. Score plot obtained in the principal component analysis, where we can appreciate a good separation of the two groups of study.

component analysis, where we can appreciate a good separation of the two groups of study.

Disease Models & Mechanisms 11: doi:10.1242/dmm.033423: Supplementary information

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Figure 2. Histology of the aortic valves control rabbits. It is shown myosin regulatory light chain 2 (Myl2, A),myosin light

tropomyosin alpha-1 chain (TPM-1, C), L-lactate dehydrogenase B chain (LDHB, D) and calreticulin (CALR, E) staining (arrow). Scale bar

corresponds to 1mm in 20x images and 100µm in 200x images. *: Aortic valve.

Figure 2. Histology of the aortic valves control rabbits. It is shown myosin regulatory light chain 2 (Myl2, A),myosin light

lactate dehydrogenase B chain (LDHB, D) and calreticulin (CALR, E) staining (arrow). Scale bar

corresponds to 1mm in 20x images and 100µm in 200x images. *: Aortic valve.

Figure 2. Histology of the aortic valves control rabbits. It is shown myosin regulatory light chain 2 (Myl2, A),myosin light chain 3 (Myl3, B),

lactate dehydrogenase B chain (LDHB, D) and calreticulin (CALR, E) staining (arrow). Scale bar

Disease Models & Mechanisms 11: doi:10.1242/dmm.033423: Supplementary information

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