Deletion of CD73 in mice leads to aortic valve dysfunction

    Deletion of CD73 in mice leads to Aortic Valve Dysfunction P Zukowska, B Kutryb-Zajac, A. Jasztal, M Toczek, M Zabielska, T Borkowski, Z Khalpey, RT Smolenski, EM Slominska PII: DOI: Reference:

S0925-4439(17)30049-2 doi:10.1016/j.bbadis.2017.02.008 BBADIS 64687

To appear in:

BBA - Molecular Basis of Disease

Received date: Revised date: Accepted date:

1 August 2016 6 February 2017 8 February 2017

Please cite this article as: P. Zukowska, B. Kutryb-Zajac, A. Jasztal, M. Toczek, M. Zabielska, T. Borkowski, Z. Khalpey, R.T. Smolenski, E.M. Slominska, Deletion of CD73 in mice leads to Aortic Valve Dysfunction, BBA - Molecular Basis of Disease (2017), doi:10.1016/j.bbadis.2017.02.008

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Deletion of CD73 in mice leads to Aortic Valve Dysfunction Zukowska P, 1Kutryb-Zajac B, 2Jasztal A, 1Toczek M, 1Zabielska M, 1Borkowski T, 3 Khalpey Z, 1Smolenski RT, 1Slominska EM

T

1

Department of Biochemistry, Medical University of Gdansk; 2Jagiellonian Center for Experimental Therapeutics, Jagiellonian University, Krakow, Poland; 3Department of Surgery, Division of Cardiothoracic Surgery, University of Arizona, College of Medicine, Tuscon

SC R

IP

1

NU

Corresponding Author:

TE

D

MA

Dr Ewa M. Slominska, Department of Biochemistry, Medical University of Gdansk, 80-211 Gdansk, Debinki 1, Poland , Phone: +48 58 349 1464, Fax: +48 58 3491465, e-mail: [email protected]

Running Title: Aortic valve dysfunction in CD73 knock-out mice.

CE P

Word Count: 8 223

AC

Abstract: 249 Abbreviations: 91 Manuscript: 5650 Figure legends: 413 References: 1645

1

ACCEPTED MANUSCRIPT ABSTRACT

Aortic stenosis is known to involve inflammation and thrombosis. Changes in activity of

T

extracellular enzyme - ecto-5'-nucleotidase (referred also as CD73) can alter inflammatory

IP

and thrombotic responses. This study aimed to evaluate the effect of CD73 deletion in mice

SC R

on development of aortic valve dysfunction and to compare it to the effect of high-fat diet. Four groups of mice (normal-diet Wild Type (WT), high-fat diet WT, normal diet CD73-/-, high-fat diet CD73-/-) were maintained for 15 weeks followed by echocardiographic analysis

NU

of aortic valve function, measurement of aortic surface activities of nucleotide catabolism enzymes as well as alkaline phosphatase activity, mineral composition and histology of aortic

MA

valve leaflets.

CD73-/- knock out led to an increase in peak aortic flow (1.06±0.26m/s) compared to WT (0.79±0.26m/s) indicating obstruction. Highest values of peak aortic flow (1.26±0.31m/s)

D

were observed in high-fat diet CD73-/- mice. Histological analysis showed morphological

TE

changes in CD73-/- including thickening and accumulation of dark deposits, proved to be melanin. Concentrations of Ca2+, Mg2+ and PO43- in valve leaflets were elevated in CD73-/-

CE P

mice. Alkaline phosphatase (ALP) activity was enhanced after ATP treatment and reduced after adenosine treatment in aortas incubated in osteogenic medium. AMP hydrolysis in CD73-/- was below 10% of WT. Activity of ecto-adenosine deaminase (eADA), responsible

AC

for adenosine deamination, in the CD73-/- was 40% lower when compared to WT. Deletion of CD73 in mice leads to aortic valve dysfunction similar to that induced by high-fat diet suggesting important role of this surface protein in maintaining heart valve integrity.

KEY WORDS

adenosine; ecto-5’-nucleotidase; aortic stenosis; inflammation; CD73 knock-out mice

2

ACCEPTED MANUSCRIPT ABBREVIATIONS

2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

ADA

adenosine deaminase

ADP

adenosine diphosphate

ALP

alkaline phosphatase

AMP

adenosine monophosphate

AOPCP

α,β-Methylene-ADP

AS

aortic stenosis

ATP

adenosine triphosphate

AVA

Aortic Valve Area

IP SC R NU MA

e5’NT, CD73 ecto-5’-nucleotidase

erythro-9-(2-hydroxy-3-nonyl) adenine

D

EHNA

T

ABTS

TE

eNTPD, CD39 ecto-nucleoside triphosphate diphosphohydrolase

Hank’s balanced salt solution

HE stain

Hematoxylin and eosin stain

HPLC

high performance liquid chromatography

LC/MS

liquid chromatography – mass spectrometry

LVEF

Left Ventricular Ejection Fraction

AC

MCSF

CE P

HBSS

Macrophage Colony-Stimulating Factor

MMPs

matrix metalloproteinases

TAOS

total antioxidant status

TGF-ß1

transforming growth factor ß1

TNF-α

tumor necrosis factor

VICs

valvular interstitial cells

WT

Wild Type

3

ACCEPTED MANUSCRIPT 1. INTRODUCTION

Aortic valve disease is a major cause of a morbidity and mortality. The prevalence of aortic

T

stenosis is estimated to be 0.5% in the general population and is much higher (2%-7%) in

IP

individuals over 65 years of age (1). The inflammation is thought to have a key role in the pathophysiology of aortic stenosis (AS), regardless of the presence of anatomical

SC R

abnormalities (2). Early changes that result in aortic stenosis are related to an active inflammatory process involving infiltration of T lymphocytes and monocytes, as well as lipid and calcium deposition and basement membrane damage (3). Damaged valves demonstrate

NU

the presence of mast cells and increased levels of pro-inflammatory cytokines: Il-1ß, tumor necrosis factor (TNF-α) and transforming growth factor ß1 (TGF-ß1) (4). The disturbances

MA

occurring in aortic valve leaflets are characterized by an excessive expression of matrix metalloproteinases (MMPs), which affect the extracellular matrix remodeling and development of local mineralization outbreaks (5). Monocytes migrate to the subendothelial

D

space and differentiate into macrophages. Activated macrophages produce pro-osteogenic

TE

cytokines, growth factors and proteolytic enzymes. The remodeling of an extracellular matrix, thickening and stiffening of leaflets as a result of proteolytic activity lead to valve dysfunction

CE P

(6).

Nucleotides and their metabolites present in the extracellular space play an important role in the moderating of an inflammatory process pathogenesis (7). Extracellular nucleotides are

AC

important signal molecules, acting via receptors on a cell surface (8). There are two major families of purine receptors - P1 for nucleosides and P2 for nucleotides. The activation of P2 receptors results in a stimulation of signal transduction pathways, which most commonly lead to a response opposite to that induced by P1 receptor activation (9). Extracellular nucleotides stimulate the inflammation, while their catabolites attenuate it (10). Adenine nucleotides and nucleosides are degraded by cell surface ecto-enzymes. The extracellular ATP is hydrolized to AMP by phosphatases, such as CD39 (ecto-nucleoside triphosphate diphosphohydrolase 1, eNTPDase 1). Then, the extracellular AMP could be converted to adenosine by CD73 (ecto5'-nucleotidase, e5'NT) (11) or alkaline phosphatase (ALP), but CD73 has been hypothesized to be the primary enzyme capable to the extracellular AMP hydrolysis (12). The adenosine is converted to inosine via ecto-adenosine deaminase (eADA) (13). The extracellular adenosine, produced by CD73, demonstrates antithrombotic and vasodilatory properties (14). It is a ligand for the four G protein-coupled receptor subtypes - A1, A2A, A2B and A3. Each of these receptors acts via various signal transduction mechanisms (15). The high expression of 4

ACCEPTED MANUSCRIPT P1 receptors on immune cells indicates their important role in the immune response regulation (16, 17). Some studies have shown a high level of the ecto-nucleotidase expression in stenotic aortic valves. It was also demonstrated, that the adenosine promotes mineralization in valve

T

interstitial cells (VICs) cultures and the expression of osteoblastic genes (18). Conversely, in

IP

patients with a NT5E gene mutation encoding CD73, an increased vascular calcification was observed (19). The enzymatic activity of CD73 was absent in fibroblasts of family members

SC R

affected by this mutation, which confirms the importance of CD73 in the inhibition of an ectopic tissue calcification (20). Therefore, attempting to clarify the exact role of the CD73 activity changes in the pathophysiology of valvular disease seems justified. The study aimed

NU

to investigate the impact of CD73 activity disturbances on the function, structure and metabolism of a murine aortic valve. The changes were compared to the effects of a high fat

MA

diet, which is one of the established experimental models of aortic valve disease (21-23). Therefore, we used WT mice fed the high-fat diet as a reference group for the development of aortic stenosis. The CD73 -/- mice on the high-fat diet were analyzed to test if the CD73

TE

D

deletion will lead to the exacerbation of an already existing aortic valve dysfunction.

2. MATERIALS AND METHODS

CE P

2.1. Animal maintenance and experimental protocol

All experiments were performed with approval of the local Bioethical Committee at the

AC

Medical University of Gdansk. C57BL/6J CD73-/- mice were obtained from Heinrich-HeineUniversität in Düsseldorf, Germany (24). Eighty four C57BL/6J Wild Type (WT) and C57BL/6J CD73-/- (CD73-/-) mice were used for these experiments. Each mouse was housed in an individually ventilated cage (23±1 ºC, 40±10% humidity) with a 12/12 h light/dark cycle. At the age of 9 weeks, the animals were randomly divided into: normal-fat diet WT (n=21), normal-fat diet CD73-/- (n=21), high-fat diet WT (n=21) and high-fat diet CD73-/(n=21). The high-fat diet contained 60% kcal fat (D12492) while the normal-fat diet contained 10% kcal fat (D12450B, both from Research Diet, New Brunswick, USA). The diet treatment was maintained for 15 weeks. Once a week, the body weight and daily food intake were monitored. At the end of the diet treatment, 6-month-old mice were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) intra-peritoneally (i.p). Subsequently, animals underwent a two-dimensional echocardiography with aortic Doppler ultrasound. After echocardiographic analysis, blood and plasma samples were collected and immediately frozen in liquid nitrogen. Then, after opening the chest, hearts and aortas were removed. The aortic 5

ACCEPTED MANUSCRIPT valve was excised and placed in 4% formalin, and the aortas in physiological saline. For detailed analysis of valve mineral deposits, in additional experiments, CD73-/- (n=24) and WT (n=24) mice, fed standard chow diet, were used. At the age of 1, 3, 6 and 12 months,

T

mice were subjected to anesthesia and aortic valve with a part of the ascending aorta were

IP

collected.

SC R

2.2. Echocardiographic Examination

Mice were anesthetized with ketamine and xylazine, as described earlier. After chest hair

NU

removal, animals were placed on a heated platform to maintain the body temperature at 37ᵒ C. The transthoracic echocardiography was performed with the SONOLINE Sienna Ultrasound Imaging System (Siemens, UK). The probe (12 MHz VF13-5) was placed over the anterior

MA

chest wall and directed towards the ascending aorta in 2D mode, which was then switched to Doppler flow velocity mode. The readings were recorded and used directly or applied for

D

Aortic Valve Area (AVA) and Left Ventricular Ejection Fraction (LVEF) calculation. AVA was measured by continuity equation. LV end-diastolic volume (LVEDV) and LV end-

TE

systolic volume (LVESV) were acquired and LVEF was calculated from the formula LVEF[%] = ((LVEDV-LVESV)/LVEDV) x 100. Hemodynamic parameters, including Stroke

CE P

Volume (SV), Heart Rate (HR) and Cardiac Output (CO) were collected. Doppler flow velocity measurements were calibrated by a comparison with readings obtained with the high

AC

resolution ultrasound system (Vevo 1100, VisualSonics Inc, Canada) in the same animal. 2.3. Aortic wall extracellular catabolism of adenine nucleotides

The thoracic and abdominal aorta were harvested from CD73-/- and WT mice. These were rinsed with 0.9% NaCl and dissected from the surrounding tissues. Thoracic and abdominal aortic sections were cut longitudinally to expose the endothelial surface and analyzed for the activities of extracellular adenine nucleotide catabolism enzymes as previously described (25). Aortic sections were placed in wells of the 24-well plates with 1 ml of Hanks Balanced Salt Solution (HBSS). HBSS buffer at pH 7.35 was composed of 1.3 mM CaCl2 x 2H2O, 5.4 mM KCl, 0.4 mM KH2PO4, 0.8 mM MgSO4 x 7H2O, 0.14 M NaCl, 0.1 mM NaH2PO4 x 7H2O and 4.2 mM NaHCO3. Before an incubation, 5.6 mM glucose was added. The aortas were pre-incubated at 37 °C for 15 min. Substrates, appropriate for each extracellular enzyme, were sequentially added to the medium: 50 µM adenosine triphosphate (ATP) for ectonucleoside triphophate diphosphohydrolase (eNTPD), 50 µM adenosine monophosphate 6

ACCEPTED MANUSCRIPT (AMP) for ecto-5’-nucleotidase (e5’NT) and 50 µM adenosine for ecto-adenosine deaminase (eADA). After 0, 5, 15 and 30 min of the incubation (37°C), the samples of 50 µl were collected. Following the incubation with each substrate, the medium was removed and

T

replaced by the fresh one. During the determination of ATP and AMP hydrolysis rates, an

IP

adenosine deaminase inhibitor – EHNA (erythro-9-(2-hydroxy-3-nonyl) adenine) at a concentration of 5 µM was added to the buffer. Before the analysis, samples were centrifuged

SC R

(20800g/ 10 min/ 4 ºC). The conversion of the substrates into the products was measured by high performance liquid chromatography (HPLC) as previously described (26). The reaction rates were normalized to aorta surface area estimated using the ImageJ Software. Data are

NU

shown in nmol/min/cm2.

The contribution of alkaline phosphatase (ALP) activity in the vascular AMP hydrolysis on

MA

the aortic surface of CD73-/- and WT mice was also evaluated. As described above, the aortic sections were placed in wells of the 24-well plates with 1ml of HBSS with 5.6 mM glucose. After a preincubation with appropriate inhibitors: 5 µM EHNA (adenosine deaminase

D

inhibitor) and 500 µM Levamisole or 2 mM L-Cysteine (alkaline phosphatase inhibitors) (27)

TE

(28), 50 µM AMP was added. The samples were collected in 0, 5, 15 and 30 minute of the incubation (37ᵒ C). WT aortic sections were also incubated in the presence of 50 µM α,β-

CE P

Methylene-ADP (AOPCP) - an inhibitor of the CD73 activity and 500 µM Levamisole to assess the participation of particular enzyme activities: e5'NT and ALP in the vascular AMP hydrolysis. The conversion of AMP into adenosine was measured by the HPLC. Data are

AC

shown in nmol/min/cm2.

2.4. Effect of ATP and Adenosine treatment on Alkaline Phosphatase activity in aortic calcification model

Fragments of the CD73-/- aortic root, including the aortic valve, ascending aorta and aortic arch were incubated for 72 hours in an osteogenic media supplemented with 4 mmol/L Lglutamine, 100 U/ml penicillin, 100 µmol/l streptomycin, and 10% fetal calf serum, as well as ascorbate-2-phosphate (50 µg/ml), dexamethasone (10 nmol/l) and B-glycerol phosphate (10 mmol/L) (all purchased from Lonza). Additionally, the aortas were treated with ATP (100 µmol/l added every 24 h) and adenosine (50 µmol/l) in the presence of EHNA (5 µmol/l). ALP activity in the aortic fragments was determined colorimetrically by using alkaline phosphatase assay kit (Abcam; ab83369) according to the manufacturer's instructions. The

7

ACCEPTED MANUSCRIPT enzyme activity was expressed as units per mg of protein. One unit of the activity was defined as the amount of enzyme that hydrolyses 1 µmol substrate per min.

T

2.5. Determination of the Ca2+, Mg2+ and PO43- concentration in aortic valves

IP

Analytical methods were adopted from previous studies (29, 30). The aortic root portions,

SC R

including the aortic valve, ascending aorta and aortic arch were placed in 6 M HCl (weight/volume ratio 1:3) for 12 h at room temp. and then centrifuged (30 min/ 2060g/ 21 ºC). To determine the Ca2+ concentration the supernatant was diluted 62 times with distilled water.

NU

20 µl of the sample and 200 µl of 200 µM Arsenazo III in acetate buffer, pH 5.6 were loaded into the 96-well plate and incubated for 5 min at room temp. The absorbance was read at 630 nm. To determine the Mg2+ concentration the supernatant was diluted 62 times with distilled

MA

water. 100 µl of the sample and 100 µl of the mixture containing 200 µM Calmagite, 200 µM EGTA and 200 µM triethanoloamine in the ammonia buffer, pH 10.1 were loaded into the 96well plate and incubated for 5 min at room temp. The absorbance was read at 490 nm. To

D

determine the PO43- concentration the supernatant was diluted 62 times with 0.6 M H2SO4.

TE

200 µl of the sample and 40 µl of 14.2 mM ammonium molybdate were loaded into the 96well plate. The plate was shaken for 10 min. To each well 40 µl of the mixture containing

CE P

1mM malachite green, 3.5g/l polyvinyl alcohol in 6 mM H2SO4 was added. Then the plate was shaken for 20 min and the absorbance was read at 630 nm.

AC

2.6. Histological evaluation of aortic valves

The calcium deposits, elastic fibers, fibrin clots and the extracellular matrix accumulation were evaluated in aortic valve leaflets. Fragments of hearts containing the aortic valve were dissected and fixed with 4% paraformaldehyde for 24 h, dehydrated and then embedded in paraffin wax blocks. Subsequently, 5 µm sections were dissected from the blocks, which were placed on the microscope slides and secured with the coverslips. The slides were then stained with either Alizarin Red, Orcein Stain and Martius Scarlet Blue Stain, using the specific protocols for each stain, which is shown in the Table 1. Good-quality leaflet sections of each group were selected and the leaflet area and thickness were measured using an image analysis software (ImageJ 1.48v). Results of the morphometric analysis were performed as the average leaflet thickness and leaflet area ratio.

8

ACCEPTED MANUSCRIPT To bleach the melanocytes, the sections were incubated with heated (60 ᵒ C, 10 min) 4% H2O2 in PBS (pH 7.6) for 1 h. Subsequently, the sections were washed in running tap water for 3 min. The slides were then stained with Hematoxylin and eosin (HE stain).

IP

T

2.7. Determination of blood adenosine concentration

SC R

Blood samples were collected from the jugular vein. To determine the adenosine level, the samples were immediately frozen in a liquid nitrogen, extracted with an acetonitrile (ratio 1:2.4) and centrifuged (20800g/5min/4ºC). The supernatant obtained was dried at 60°C using

NU

a rotary vacuum concentrator and the residue was dissolved in a water at a volume equal to the initial blood volume. The adenosine concentration was determined using the high performance liquid chromatography – mass spectrometry (LC/MS). The LC/MS equipment

MA

contained a Surveyor MS autosampler, quaternary Surveyor MS pump and a degasser connected to TSQ Vantage triple quadrupole mass detector. We used heated electrospray ionization in the positive mode. The column used for the chromatographic separation was the

D

Phenomenex Synergi Hydro RP 100 50 x 2mm Synergi Hydro – RP with a C18 particle size

TE

of 2.5 µm. During the separation the column temperature was maintained at 25 ºC. The mobile phase consisted of buffer A containing 0.1% formic acid in the acetonitrile and buffer

CE P

B containing 0.1% formic acid. The injection volume for the each sample at a flow rate 0.2 ml/min, was 2 µl. As an internal standard 2 – chloroadenosine was used. The adenosine and the internal standard were identified as a [M + H]+ ions based on the determined mass to

AC

charge ratio (m/z) transitions: adenosine 268 > 136.15, internal standard 302 >171.10. 2.8. Determination of plasma total antioxidant status.

The total antioxidant status (TAOS) in plasma was determined with the ABTS assay, based on the ability of the plasma to scavenge the 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid (ABTS+) radical (31). The relative inhibition of ABTS+ formation, after the plasma addition, is proportional to the antioxidant capacity of the sample. To measure the total antioxidant status in plasma, 5µl of plasma was added to the reaction mixture, containing 7 mM ABTS and 2.45 mM potassium persulfate in 100 ml pure water (pH 7.2), which was then incubated 10 min at 25 ºC in the 96-well plate. The absorbance in the test and control samples (5 µl NaCl instead of plasma) was read at 405 nm, using a BioTek microplate reader. The ABTS scavenging effect, expressed as a percentage, was calculated as follows: [E] = 100 x (Ac – 9

ACCEPTED MANUSCRIPT At)/Ac, where Ac is the control sample absorbance and At is the absorbance of the test sample.

T

2.9. Statistical analysis

IP

The results were presented as mean ± SEM. The statistical analysis was performed using

SC R

Graph Pad Prism 7 (Graph Pad Software). Two-way analysis of variance with post-hoc Tukey test was used for the intragroup comparisons. Paired and unpaired Student t test were used for

NU

comparisons between two groups. A p-value < 0.05 was considered a significant difference.

3. RESULTS

MA

The AMP hydrolysis rate in the CD73-/- mice was approximately ten times lower in comparison with the WT (Figure 1B), which was consistent with the CD73 gene deletion. The

D

high-fat diet did not cause a statistically significant change in the AMP hydrolysis on the

TE

aortic surface. The ATP hydrolysis rate in the CD73-/- mice fed a normal-fat diet was approximately 25 % higher compared to control mice fed the same diet. A similar difference

CE P

was demonstrated between the CD73-/- and WT group on a high-fat diet. Decreased ATP hydrolysis was also shown in the high-fat diet group (Figure 1A). There was a significantly reduced adenosine deamination rate on the surface of CD73-/- aorta irrespective of the diet as

AC

compared to WT (Figure 1C).

The participation of alkaline phosphatase (ALP) activity in total vascular AMP hydrolysis on the aortic surface of CD73-/- and WT was analyzed. After using the Levamisole, the level of AMP hydrolysis decreased by approx. 35% in WT. On the aortic surface of CD73-/- mice, the AMP hydrolysis was almost completely inhibited (Figure 2A). The contribution of e5'NT and ALP activities in the vascular AMP hydrolysis was also tested in WT. The vascular AMP hydrolysis decreased by about 35% following the inhibition of ALP activity, while after the inhibition of CD73 activity - by approx. 90%. The rate of AMP hydrolysis was less than 4% of control, when both of activities were inhibited (Figure 2B). These results were also confirmed, when the ALP activity was inhibited by the L-cysteine – an another known ALP inhibitor (Supplemental Figure S1). To further characterise the implications of a lack of CD73 activity for the aortic valve function, we conducted two-dimensional echocardiographic measurements and examined the 10

ACCEPTED MANUSCRIPT aortic valve flow velocity using Doppler ultrasound. The peak aortic valve flow was significantly increased in CD73-/- mice fed both, a normal-fat and high-fat diet in comparison with control animals (Figure 3A). Furthermore, the high-fat diet group had a considerable

T

increase in the aortic valve flow velocity in both groups of mice involved in the experiment

IP

(CD73-/- and WT). The highest peak aortic valve flow was noted in the CD73-/- group fed a high-fat diet (Figure 3A). Aortic Valve Area (AVA) was significantly smaller in the CD73-/-,

SC R

regardless of the the diet treatment, compared to WT. The high-fat diet caused a substantial decrease in AVA of control animals (Figure 3B). The calculated Left Ventricular Ejection Fraction (LVEF) was not different between WT and CD73-/- mice on a normal or high fat

NU

diets (Figure 3C). The in vivo analysis of the hemodynamic parameters in mice revealed no significant differences (Supplemental Table S1). The measurements of HR and CO did not

MA

differ between CD73-/- and WT.

We further evaluated whether the lack of CD73 activity had an impact on the mineralization of the aortic valves. The concentration of Ca2+, Mg2+ and PO43- in the 1-, 3-, 6- and 12-month-

D

old CD73-/- and WT excised aortic valve and the aortic arch were measured. The level of

TE

Ca2+, Mg2+ as well as PO43- was significantly higher in the aortic valve leaflets of CD73-/mice compared to WT, irrespective of their age (Figure 4C). The Ca2+ concentration increased

CE P

with age in both, CD73-/- and WT mice. The highest increase in Ca2+ concentration was observed between 3- and 6-month old CD73-/- mice (Figure 4C). Age did not affect changes in the Mg2+ and PO43- concentration in the aortic valve leaflets of the mice involved in the

AC

experiment (CD73-/- and WT).

The treatment of CD73-/- mice aortic root portions with the osteogenic media and ATP (100 µmol/l) caused significant increase in the ALP activity in the aortas compared with the tissue treated with the osteogenic media only after 72 hours (Figure 5). The treatment of CD73-/aortic root portions with the osteogenic media and the adenosine (50 µmol/l) caused considerable decrease in the ALP activity compared with both, the ATP treated tissue and tissue treated with the osteogenic media only after 72 hours (Figure 5). To test the aortic valve structure and morphology, the histological analysis was performed. CD73-/- aortic valves were identified because they had a visible leaflet thickening in comparison to WT (Figure 4A and 4B). The high-fat diet also resulted in a leaflet thickening in WT aortic valves. Furthermore, we observed an increased aortic valve thickening and enlargement in CD73-/- mice when exposed to a high fat diet (Figure 4A and 4B). We noticed that there was an intensified accumulation of black-brown deposits on the valve leaflets which 11

ACCEPTED MANUSCRIPT was consistent throughout the valve; this proved to be melanin. There was a substantial amount of valvular deposits found in the aortic valves of mice that were on the high-fat diet. The CD73-/- mice aortic valves on a high-fat diet were characterized by a larger amount of

T

melanin deposits compared to WT mice on the same diet. In order to standardize our

IP

observations, a valve thickening is presented as the average aortic valve leaflets thickness to the leaflets area ratio. The ratio was significantly increased in the CD73-/- mice (Figure 4B).

SC R

The area occupied by the melanin deposits is significantly higher in the CD73-/- mice compared to WT. The highest values of the area covered by the deposits were observed in the CD73-/- mice on a high-fat diet. The histological analysis showed no calcium deposits,

NU

excessive accumulation of amorphous matrix and fibrin clots in the analyzed valve leaflets, probably because of the lower sensitivity of this type of examination compared to the

MA

chemical analysis.

We also investigated the total antioxidant status (TAOS) in plasma and blood adenosine concentration of the CD73-/- and WT mice, fed a normal-fat diet (Figure 6). The adenosine

D

level in the CD73-/- mice blood was significantly lower compared to the WT (Figure 6A).

TE

The total antioxidant status was approximately 25% lower in comparison to WT (Figure 6B).

CE P

After the experiment, the CD73-/- normal-fat and high-fat diet mice, had a lower body weight compared to WT. Both WT and CD73-/- high-fat diet groups achieved a higher weight gain than mice fed a normal-fat diet (Supplemental Figure S2A). To investigate whether the changes in the extracellular adenosine level have an impact on the carbohydrate and lipid

AC

metabolism, we measured a fasting glucose level in our animals. Following 15 weeks of feeding the high-fat diet, CD73-/- had a significantly higher concentration of blood glucose compared to WT. In the case of both, CD73-/- and WT, the high-fat diet caused an increase in blood glucose concentration (Supplemental Figure S2B). The CD73-/- mice on a normal-fat diet were characterized by the considerably lower total cholesterol, HDL and LDL levels, and in turn, higher TG level compared to a normal-fat diet WT. The high-fat diet caused increased levels of total cholesterol, LDL and TG and decreased level of HDL in WT. In CD73-/- fed the high-fat diet, levels of total cholesterol, TG, but also HDL were higher, compared to the normal-fat diet (Supplemental Figure S2C).

4. DISCUSSION

12

ACCEPTED MANUSCRIPT This study is the first to demonstrate that vascular CD73 activity is essential for the normal aortic valve function and the preservation of its structure. The deletion of this activity in CD73-/- mice results in the aortic valve leaflet thickening, mineralization and functional

T

impairment of the valve consistent with stenosis.

IP

4.1. Changes in vascular activities of CD73 and related enzymes in CD73-/- mice.

SC R

The CD73 gene deletion resulted in the almost complete loss of pathway to degrade the AMP to the adenosine at a vascular surface. This indicates that CD73 is the most important enzyme of this process and other activities such as alkaline phosphatase play only a minor role. The

NU

lack of the CD73 activity in CD73-/- mice had some effect on the activities of other extracellular nucleotide metabolism enzymes on the vascular surface. The ecto-nucleoside

MA

triphosphate diphosphohydrolase 1 (eNTPD, CD39) activity was slightly higher in CD73-/compared to WT, while the ecto-adenosine deaminase (eADA) activity was lower. These enzymes, together with CD73 are elements of the extracellular ATP breakdown cascade, that

D

is mainly located on the endothelial surface (11, 32). These changes in eNTPD and eADA

TE

could be a consequence of decreased formation of the extracellular adenosine by CD73. However, changes in the eNTPD and eADA activities were relatively minor and could not

CE P

compensate for the loss of CD73 activity. Consistent with the results in CD73-/- mice, the AMP hydrolysis rate on the aortic surface decreased only slightly following application of an inhibitor of the alkaline phosphatase

AC

(ALP), while after an inhibition of CD73 decreased more than 90% as compared to control. This further indicates that the ALP contribution is minor as compared to CD73, which is the predominant enzyme converting the AMP to adenosine on the aortic surface. Some discrepancy between effect of the CD73 gene delection and application of the ALP inhibitor may be a consequence of an eventual ALP inhibitors effect on CD73 activity. The Km values of CD73 for AMP is in the low micromolar range (33) that is much lower (few orders of magnitude) than ALP Km values for AMP. In some tissues Vmax for ALP could be higher (but only few times) compared to 5'NT (34). However, considering that extracellular concentrations of AMP do not exceed micromolar, even under severe stress, CD73 seems to play more important role in AMP degradation than ALP (35). Only under artificial conditions with millimolar vascular AMP concentrations degradation via ALP could prevail.

13

ACCEPTED MANUSCRIPT Consequences of CD73 deletion on aortic valve function The lack of CD73 activity was associated with a significant increase in the peak blood flow velocity through the aortic valve and considerably smaller AVA. We found the LVEF to be

T

normal in the CD73 mutants, suggesting that disturbances of aortic blood flow velocity result

IP

from aortic valve dysfunction itself. It is consistent with clinical data indicating, that LVEF is

SC R

normal in most patients with AS (24, 36). These results are highly suggestive of the aortic stenosis. The change in CD73-/- peak aortic flow was similar to increase in the peak aortic flow induced by the high-fat diet. The latter is an established model of the aortic valve disease in C57BL/6 mice (21) with the abnormalities closely resembling those found in the early

NU

phases of the aortic stenosis in humans. Another study demonstrated severe inflammation in the aortic valves of ApoE-/- mice fed a Western Diet, that led to the calcific aortic valve

MA

disease (37). This study showed strong MMPs activation in the aortic valves resulting in valve stenosis and a disturbed blood flow. Our findings seem to be consistent with these studies. The disrupted CD73 activity and the decreased adenosine levels created a pro-inflammatory

TE

D

environment in vessels including the aortic valve. 4.2. Increased mineralization and ALP activity in CD73-/- aortic valves

CE P

The chemical analysis showed a significant increase in the Ca2+, Mg2+ and PO43- in the aortic valve leaflets, ascending aortas and aortic arches of CD73-/- mice as compared to WT. The calcium and phosphate content increased in an age-dependent manner. The tissue

AC

mineralization is a consequence of local and general factors including an inflammation. The loss of the anti-inflammatory properties of the CD73-derived adenosine may lead to an activation of these processes. One such mechanism is an activation and differentiation of valvular interstitial cells (VICs) into an osteoblast-like phenotype (38). The earlier studies demonstrated that the CD73-/- mice are prone to an ectopic calcification leading to the stiffening of the joints and mineralization. The CD73-/- mice were also characterized by a significantly increased phosphate concentration in the serum and a reduced pyrophosphate concentration in plasma, resulting in a pro-mineralizing environment (39). Contrary to our findings, a recently published work indicated that the A2A receptor activation could lead to a valve mineralization and dysfunction (18). This study also suggested that the increased expression and activity of CD73 contributes to the aortic valves calcification. One reason that could lead to this conclusion is the method for the CD73 activity measurements, that was based on an inorganic phosphate release from the AMP. It is prone to interference by release of phosphate from other than AMP sources and cannot differentiate from other phosphatases. 14

ACCEPTED MANUSCRIPT The discrepancy with our conclusions could be a consequence of using only selected areas of the pathologically altered valves in this study. Our earlier research demonstrated that an expression of CD73 varies in different regions of the pathological valve leaflets (40). It was

T

significantly decreased in most areas with the exception of region around the calcium

IP

deposits, where the CD73 expression was maintained. The treatment of CD73-/- mice aortic sections with an osteogenic media and ATP enhanced the ALP activity in the aortas, whereas

SC R

the adenosine treatment reduced the ALP activity induced by an osteogenic media. An earlier study of our group is consistent with these findings (38). Primary cultures of human valve interstitial cells (VICs) treated with ATP or ATP-γ-S (agonist of the P2Y receptor) could be

NU

transformed into the osteoblasts (as evidenced by an increase in alkaline phosphatase activity) in a similar manner as found after the treatment with an osteogenic media. Furthermore, VICs

MA

treatment with the adenosine did not cause an increase in ALP activity in VICs, while the adenosine treatment together with an osteogenic medium lead to a decreased ALP expression and activity. We have measured the ALP activity as an indicator of the transformation of

D

valve or vascular cells into a pro-calcific phenotype. While our experiment with aortic

TE

fragments in the osteogenic media did not directly provide the evidence for control of calcification by ATP or adenosine, many studies indicated direct relation of ALP with

CE P

calcification. It is well known, that ALP plays a key role in the calcification process in the valves. It is also known that ALP activity inhibition prevents the formation of osteonectin expression and calcified nodules in VICs grown in an osteogenic media (41). Recent study of St. Hilaire C et al. published as an abstract on the arterial calcification due to deficiency of

AC

CD73 indicated the vascular calcification development in immunocompromised transgenic mice following exposure to induced pluripotent stem cells (iPSCs) from CD73 deficient patients (42). This findings confirmed direct association of CD73 with the ectopic calcification. Nevertheless, further studies are required to clarify the exact role of CD73 and the mechanism in the pathogenesis of aortic valve calcification. 4.3. Morphological changes of CD73-/- aortic valves The changes in the aortic valve morphology are consistent with the aortic valve dysfunction. Our histological images of the aortic valves suggest the presence of the thickened leaflets in CD73-/- mice. We noticed that the CD73-/- mice had a lot of black-brown stained deposits on the valves and within the vicinity of these deposits the valve walls tended to be thickened. The accumulated deposits increased in the high-fat diet animals. More detailed studies have proven that the pigment in these valves is the melanin (discoloration on hydrogen peroxide 15

ACCEPTED MANUSCRIPT exposure, data not shown). Morphometric measurements of the valve leaflets thickness and the area and amount of melanotic deposits highlight, that the structural and functional abnormalities occur in CD73-/-, particularly if fed a high-fat diet. It is important to note that

T

the popular Von Kossa staining used frequently in the literature stains calcium deposits black

IP

(43). This can lead to a misrepresentation as the pre-existing black deposits could be interpreted as a positive effect of this stain. Thus, in order to verify our results, we used the

SC R

Alizarin Red method, which stains the calcium deposits an intensive red color. It excluded the presence of calcium deposits in the examined aortic valves. Balani K et al. have shown, that melanocytes are involved in the valve reconstruction and remodeling. They have also shown,

NU

that mice tricuspid valve pigmentation is associated with the accumulation of melanocytes, which affects its stiffness (44). The presence of melanocytes affects the viscoelastic properties

MA

of the mice atrioventricular valves and it is not irrelevant to their proper functioning in the organism. Missing evidence of the mice aortic valve calcification in histological stainings may result from a different anatomical structure of the mouse aortic valves in comparison to

D

that of humans. Mice do not have the trilayer aortic valve tissue morphology characteristic of

TE

human or rabbit and pig valves (45).

CE P

4.4. Systemic metabolic disturbances in CD73-/- mice Blood adenosine concentration in CD73-/- mice was considerably lower than in WT, regardless of the diet type. Xaus J et al. have shown, that the adenosine inhibits a macrophage proliferation, which is dependent on Macrophage Colony-Stimulating Factor (MCSF) (46).

AC

The adenosine is also involved in an inhibition of TNF-α from NK cells stimulated with IL-2 (47). These adenosine - dependent mechanisms of the inflammatory reaction and immune response regulation allow us to suggest, that the decrease in the adenosine availability may have contributed to the exacerbated inflammation in the aortic valves and vessels (48, 49). Processes that contribute to the majority of vascular pathologies, such as inflammation, endothelial dysfunction and cell migration are closely related with oxidative stress (50). One of the key relevant parameters is plasma total antioxidant status (TAOS). In CD73-/- mice, plasma TAOS is lower, suggestsing an increase in the amount of free radicals and hence oxidative stress. It can be caused by both mechanisms, increased production or decreased removal of free radicals. Some studies indicate enhanced production of free radicals in inflammatory diseases, e.g. type 2 diabetes (51). Other studies linked the CD73 activity and the adenosine formation with an antioxidant effect (52). However, the exact mechanism for lowered total antioxidant status in CD73-/- mice requires further studies. 16

ACCEPTED MANUSCRIPT We have also shown that CD73-/- mice are characterized by a lower weight than WT mice. Additionally, even under the high-fat diet influence, the weight gain in CD73-/- mice was about 30% lower than the control group on the same diet. Furthermore, the CD73-/- mice

T

have an elevated blood glucose concentration and altered lipid profile compared to WT. This

IP

is consistent with earlier data (53). A study on the impact of the adenosine on the adipose tissue demonstrated that CD73-/- mice were characterized by a significantly less weight gain

SC R

and a lower content of white adipose tissue, as well as the increased free fatty acids and triglycerides in the serum (53). Reduced adenosine levels are associated with a significant increase in the hydrolysis of triglycerides to glycerol and free fatty acids. High levels of free

NU

fatty acids may contribute to the hepatic glucose production and reduced insulin sensitivity by the inhibition of glucose uptake (54).

MA

4.5. Summary

In summary, this study provides the first experimental evidence that the CD73 deletion leads

D

to the development of the aortic valve dysfunction in mice. This effect is similar to that

TE

induced by a high-fat diet and is evidenced by the structural and functional changes consistent with aortic stenosis. This indicates that CD73 plays an essential role in the aortic valve

CE P

homeostasis. CD73-/- mice may be a good model to study mechanisms and protective strategies in valvular disease.

AC

5. FUNDING

This study was supported by European Union from the resources of the European Regional Development Fund under the Innovative Economy Programme (grant coordinated by JCETUJ,

No

POIG.01.01.02-00-069/09),

National

Science

Centre

of

Poland

(2011/01/B/NZ4/03719 and 2015/19/N/NZ1/03435) and Foundation for Polish Science (TEAM/2011-8/7).

17

ACCEPTED MANUSCRIPT 6. REFERENCES 1.

Nkomo VT, Gardin JM, Skelton TN, Gottdiener JS, Scott CG, Enriquez-Sarano M.

Mohler ER. 2000. Are atherosclerotic processes involved in aortic-valve calcification? Lancet

IP

2.

T

2006. Burden of valvular heart diseases: a population-based study. Lancet 368:1005-1011.

356:524-525.

Towler DA. 2013. Molecular and cellular aspects of calcific aortic valve disease. Circ Res

SC R

3.

113:198-208. 4.

Rajamannan NM, Gersh B, Bonow RO. 2003. Calcific aortic stenosis: from bench to the

5.

NU

bedside--emerging clinical and cellular concepts. Heart 89:801-805. Rajamannan NM, Subramaniam M, Rickard D, Stock SR, Donovan J, Springett M, Orszulak T, Fullerton DA, Tajik AJ, Bonow RO, Spelsberg T. 2003. Human aortic valve

6.

MA

calcification is associated with an osteoblast phenotype. Circulation 107:2181-2184. New SE, Aikawa E. 2011. Molecular imaging insights into early inflammatory stages of arterial and aortic valve calcification. Circ Res 108:1381-1391. Jalkanen J, Yegutkin GG, Hollmén M, Aalto K, Kiviniemi T, Salomaa V, Jalkanen S,

D

7.

TE

Hakovirta H. 2015. Aberrant circulating levels of purinergic signaling markers are associated with several key aspects of peripheral atherosclerosis and thrombosis. Circ Res 116:1206-

8.

CE P

1215.

Helenius M, Jalkanen S, Yegutkin G. 2012. Enzyme-coupled assays for simultaneous detection of nanomolar ATP, ADP, AMP, adenosine, inosine and pyrophosphate concentrations in extracellular fluids. Biochim Biophys Acta 1823:1967-1975. Ferrari D, Vitiello L, Idzko M, la Sala A. 2015. Purinergic signaling in atherosclerosis.

AC

9.

Trends Mol Med. 10.

Idzko M, Ferrari D, Eltzschig HK. 2014. Nucleotide signalling during inflammation. Nature 509:310-317.

11.

Yegutkin GG. 2008. Nucleotide- and nucleoside-converting ectoenzymes: Important modulators of purinergic signalling cascade. Biochim Biophys Acta 1783:673-694.

12.

Pettengill M, Robson S, Tresenriter M, Millán JL, Usheva A, Bingham T, Belderbos M, Bergelson I, Burl S, Kampmann B, Gelinas L, Kollmann T, Bont L, Levy O. 2013. Soluble ecto-5'-nucleotidase (5'-NT), alkaline phosphatase, and adenosine deaminase (ADA1) activities in neonatal blood favor elevated extracellular adenosine. J Biol Chem 288:2731527326.

13.

Yegutkin GG. 2014. Enzymes involved in metabolism of extracellular nucleotides and nucleosides: functional implications and measurement of activities. Crit Rev Biochem Mol Biol 49:473-497. 18

ACCEPTED MANUSCRIPT 14.

Willems L, Ashton KJ, Headrick JP. 2005. Adenosine-mediated cardioprotection in the aging myocardium. Cardiovasc Res 66:245-255.

15.

Mubagwa K, Flameng W. 2001. Adenosine, adenosine receptors and myocardial protection: an updated overview. Cardiovasc Res 52:25-39. Shryock JC, Belardinelli L. 1997. Adenosine and adenosine receptors in the cardiovascular

T

16.

Vergani A, Tezza S, Fotino C, Visner G, Pileggi A, Chandraker A, Fiorina P. 2014. The

SC R

17.

IP

system: biochemistry, physiology, and pharmacology. Am J Cardiol 79:2-10.

purinergic system in allotransplantation. Am J Transplant 14:507-514. 18.

Mahmut A, Boulanger MC, Bouchareb R, Hadji F, Mathieu P. 2015. Adenosine derived

adenosine receptor. Cardiovasc Res. 19.

NU

from ecto-nucleotidases in calcific aortic valve disease promotes mineralization through A2a

St Hilaire C, Ziegler SG, Markello TC, Brusco A, Groden C, Gill F, Carlson-Donohoe H,

MA

Lederman RJ, Chen MY, Yang D, Siegenthaler MP, Arduino C, Mancini C, Freudenthal B, Stanescu HC, Zdebik AA, Chaganti RK, Nussbaum RL, Kleta R, Gahl WA, Boehm M. 2011. NT5E mutations and arterial calcifications. N Engl J Med 364:432-442. 20.

Gan XT, Taniai S, Zhao G, Huang CX, Velenosi TJ, Xue J, Urquhart BL, Karmazyn M.

D

2014. CD73-TNAP crosstalk regulates the hypertrophic response and cardiomyocyte

21.

TE

calcification due to α1 adrenoceptor activation. Mol Cell Biochem 394:237-246. Drolet MC, Roussel E, Deshaies Y, Couet J, Arsenault M. 2006. A high fat/high

855. 22.

CE P

carbohydrate diet induces aortic valve disease in C57BL/6J mice. J Am Coll Cardiol 47:850-

Hofmann B, Yakobus Y, Indrasari M, Nass N, Santos AN, Kraus FB, Silber RE, Simm A. 2014. RAGE influences the development of aortic valve stenosis in mice on a high fat diet.

23.

AC

Exp Gerontol 59:13-20.

Jung JJ, Razavian M, Kim HY, Ye Y, Golestani R, Toczek J, Zhang J, Sadeghi MM. 2016. Matrix metalloproteinase inhibitor, doxycycline and progression of calcific aortic valve disease in hyperlipidemic mice. Sci Rep 6:32659.

24.

Koszalka P, Ozüyaman B, Huo Y, Zernecke A, Flögel U, Braun N, Buchheiser A, Decking UK, Smith ML, Sévigny J, Gear A, Weber AA, Molojavyi A, Ding Z, Weber C, Ley K, Zimmermann H, Gödecke A, Schrader J. 2004. Targeted disruption of cd73/ecto-5'nucleotidase alters thromboregulation and augments vascular inflammatory response. Circ Res 95:814-821.

25.

Kutryb-Zajac B, Yuen AH, Khalpey Z, Zukowska P, Slominska EM, Taylor PM, Goldstein S, Heacox AE, Lavitrano M, Chester AH, Yacoub MH, Smolenski RT. 2016. Nucleotide Catabolism on the Surface of Aortic Valve Xenografts; Effects of Different Decellularization Strategies. J Cardiovasc Transl Res 9:119-126.

19

ACCEPTED MANUSCRIPT 26.

Smolenski RT, Lachno DR, Ledingham SJ, Yacoub MH. 1990. Determination of sixteen nucleotides, nucleosides and bases using high-performance liquid chromatography and its application to the study of purine metabolism in hearts for transplantation. J Chromatogr 527:414-420. Shioi A, Nishizawa Y, Jono S, Koyama H, Hosoi M, Morii H. 1995. Beta-glycerophosphate

T

27.

IP

accelerates calcification in cultured bovine vascular smooth muscle cells. Arterioscler Thromb

28.

SC R

Vasc Biol 15:2003-2009.

El-Aaser AA, El-Merzabani MM. 1975. Simultaneous determination of 5'-nucleotidase and alkaline phosphatase activities in serum. Z Klin Chem Klin Biochem 13:453-459.

29.

Nyman J, Ivaska A. 1995. Spectrophotometric determination of calcium in paper machine

30.

NU

white water by sequential injection analysis. Analytica Chimica Acta 308:286-292. Mikroulis D, Mavrilas D, Kapolos J, Koutsoukos PG, Lolas C. 2002. Physicochemical and

MA

microscopical study of calcific deposits from natural and bioprosthetic heart valves. Comparison and implications for mineralization mechanism. J Mater Sci Mater Med 13:885889. 31.

Wong WM, Stephens JW, Acharya J, Hurel SJ, Humphries SE, Talmud PJ. 2004. The

D

APOA4 T347S variant is associated with reduced plasma TAOS in subjects with diabetes

32.

TE

mellitus and cardiovascular disease. J Lipid Res 45:1565-1571. Kochan Z, Smolenski RT, Yacoub MH, Seymour AL. 1994. Nucleotide and adenosine

33.

CE P

metabolism in different cell types of human and rat heart. J Mol Cell Cardiol 26:1497-1503. Zimmermann H, Zebisch M, Sträter N. 2012. Cellular function and molecular structure of ecto-nucleotidases. Purinergic Signal 8:437-502. 34.

Picher M, Burch LH, Hirsh AJ, Spychala J, Boucher RC. 2003. Ecto 5'-nucleotidase and

AC

nonspecific alkaline phosphatase. Two AMP-hydrolyzing ectoenzymes with distinct roles in human airways. J Biol Chem 278:13468-13479. 35.

Borges FP, Gottardi B, Stuepp C, Larré AB, Tasca T, De Carli GA, Bonan CD. 2007. Characterization of an ecto-5'-nucleotidase (EC 3.1.3.5) activity in intact trophozoites of Trichomonas gallinae. Vet Parasitol 143:106-111.

36.

Hachicha Z, Dumesnil JG, Bogaty P, Pibarot P. 2007. Paradoxical low-flow, low-gradient severe aortic stenosis despite preserved ejection fraction is associated with higher afterload and reduced survival. Circulation 115:2856-2864.

37.

Jung JJ, Razavian M, Challa AA, Nie L, Golestani R, Zhang J, Ye Y, Russell KS, Robinson SP, Heistad DD, Sadeghi MM. 2015. Multimodality and molecular imaging of matrix metalloproteinase activation in calcific aortic valve disease. J Nucl Med 56:933-938.

38.

Osman L, Chester AH, Amrani M, Yacoub MH, Smolenski RT. 2006. A novel role of extracellular nucleotides in valve calcification: a potential target for atorvastatin. Circulation 114:I566-572. 20

ACCEPTED MANUSCRIPT 39.

Li Q, Price TP, Sundberg JP, Uitto J. 2014. Juxta-articular joint-capsule mineralization in CD73 deficient mice: similarities to patients with NT5E mutations. Cell Cycle 13:2609-2615.

40.

Kaniewska E, Sielicka A, Sarathchandra P, Pelikant-Małecka I, Olkowicz M, Słomińska EM, Chester AH, Yacoub MH, Smoleński RT. 2014. Immunohistochemical and functional

T

analysis of ectonucleoside triphosphate diphosphohydrolase 1 (CD39) and ecto-5'-

Mathieu P, Voisine P, Pépin A, Shetty R, Savard N, Dagenais F. 2005. Calcification of

SC R

41.

IP

nucleotidase (CD73) in pig aortic valves. Nucleosides Nucleotides Nucleic Acids 33:305-312.

human valve interstitial cells is dependent on alkaline phosphatase activity. J Heart Valve Dis 14:353-357. 42.

St. Hilaire C, Hui J, Huang Y, Yu Z, Yang D, Negro A, Liu Y, Davaine J-M,

NU

Schwartzbeck R, Chen G, Boehm M. In Vivo Disease Modeling Using Patient-Derived iPSCs: CD73-Deficiency Promotes Osteogenic Differentiation and Calcification. Purinergic

MA

Signaling. Cancer Immunotherapy: Immunity and Immunosuppression Meet Targeted Therapies - Abstract Book 2016: p.72. 43.

Hinton RB, Alfieri CM, Witt SA, Glascock BJ, Khoury PR, Benson DW, Yutzey KE. 2008. Mouse heart valve structure and function: echocardiographic and morphometric

D

analyses from the fetus through the aged adult. Am J Physiol Heart Circ Physiol 294:H2480-

44.

TE

2488.

Balani K, Brito FC, Kos L, Agarwal A. 2009. Melanocyte pigmentation stiffens murine

45.

CE P

cardiac tricuspid valve leaflet. J R Soc Interface 6:1097-1102. Sider KL, Blaser MC, Simmons CA. 2011. Animal models of calcific aortic valve disease. Int J Inflam 2011:364310. 46.

Xaus J, Valledor AF, Cardó M, Marquès L, Beleta J, Palacios JM, Celada A. 1999.

AC

Adenosine inhibits macrophage colony-stimulating factor-dependent proliferation of macrophages through the induction of p27kip-1 expression. J Immunol 163:4140-4149. 47.

Miller JS, Cervenka T, Lund J, Okazaki IJ, Moss J. 1999. Purine metabolites suppress proliferation of human NK cells through a lineage-specific purine receptor. J Immunol 162:7376-7382.

48.

Zernecke A, Bidzhekov K, Ozüyaman B, Fraemohs L, Liehn EA, Lüscher-Firzlaff JM, Lüscher B, Schrader J, Weber C. 2006. CD73/ecto-5'-nucleotidase protects against vascular inflammation and neointima formation. Circulation 113:2120-2127.

49.

Khalpey Z, Yuen AH, Kalsi KK, Kochan Z, Karbowska J, Slominska EM, Forni M, Macherini M, Bacci ML, Batten P, Lavitrano M, Yacoub MH, Smolenski RT. 2005. Loss of ecto-5'nucleotidase from porcine endothelial cells after exposure to human blood: Implications for xenotransplantation. Biochim Biophys Acta 1741:191-198.

50.

Fearon IM, Faux SP. 2009. Oxidative stress and cardiovascular disease: novel tools give (free) radical insight. J Mol Cell Cardiol 47:372-381. 21

ACCEPTED MANUSCRIPT 51.

Newsholme P, Cruzat VF, Keane KN, Carlessi R, de Bittencourt PI. 2016. Molecular mechanisms of ROS production and oxidative stress in diabetes. Biochem J 473:4527-4550.

52.

Le Hir M, Kaissling B. 1993. Distribution and regulation of renal ecto-5'-nucleotidase: implications for physiological functions of adenosine. Am J Physiol 264:F377-387. Burghoff S, Flögel U, Bongardt S, Burkart V, Sell H, Tucci S, Ikels K, Eberhard D, Kern

T

53.

IP

M, Klöting N, Eckel J, Schrader J. 2013. Deletion of CD73 promotes dyslipidemia and

Poncelas M, Inserte J, Vilardosa Ú, Rodriguez-Sinovas A, Bañeras J, Simó R, GarciaDorado D. 2015. Obesity induced by high fat diet attenuates postinfarct myocardial

CE P

TE

D

MA

NU

remodeling and dysfunction in adult B6D2F1 mice. J Mol Cell Cardiol 84:154-161.

AC

54.

SC R

intramyocellular lipid accumulation in muscle of mice. Arch Physiol Biochem 119:39-51.

22

ACCEPTED MANUSCRIPT FIGURE LEGEND Figure 1. CD73 knock-out profoundly decreases the rate of vascular AMP to adenosine conversion. Rate of A) ATP hydrolysis; B) AMP hydrolysis; C) Adenosine deamination on

IP

T

the surface of the CD73-/- and WT mice aorta depending following high-fat or normal-fat diet. All values are shown as mean ± SEM (n=21; Two-way ANOVA with post-hoc Tukey

SC R

test: *p<0.05; **p<0.01; ***p<0.001).

Figure 2. The CD73 inhibition but not alkaline phosphatase (ALP) inhibition most effectively blocks the vascular AMP to adenosine conversion. A) Contribution of ALP to

NU

total vascular AMP hydrolysis on the WT and CD73-/- mice aortic surface: ALP acitivity inhibited by Levamisole. B) Participation of particular enzymes: CD73 and ALP in vascular

MA

AMP hydrolysis on the WT mice aortic surface. ALP activity inhibited by Levamisole, CD73 activity inhibited by AOPCP. All values are shown as mean ± SEM (n=10; Two-way

D

ANOVA with post-hoc Tukey test: *p<0.05; **p<0.01; ***p<0.001). Figure 3. Elevated peak aortic flow and decreased AVA, with preserved LVEF in CD73-

TE

/- mice in comparison to WT. Echocardiographic parameters: A) peak aortic valve flow; B) Aortic Valve Area (AVA); C) Left Ventricular Ejection Fraction (LVEF) ; All values are

CE P

shown as mean ± SEM (n=21; Two-way ANOVA with post-hoc Tukey test: *p<0.05; **p<0.01; ***p<0.001).

AC

Figure 4. Morphological and structural changes and increased Ca2+, Mg2+ and PO43concentration in the CD73-/- aortic valves. A) histological images for normal-fat diet WT, normal-fat diet CD73-/-, high-fat diet WT and high-fat diet CD73-/-; B) the average aortic valve leaflet thickness/ leaflet area ratio and aortic valve deposits area; C) Ca2+, Mg2+ and PO43- concentration in aortic valves of 1-, 3-, 6- and 12- month – old CD73-/- and WT mice. All values are shown as mean ± SEM (n=21; Two-way ANOVA with post-hoc Tukey test and Student t test: *p<0.05; **p<0.01; ***p<0.001). Figure 5. Increase in ALP activity by ATP and decrease by adenosine treatment in CD73-/- aortic root fragments incubated in osteogenic media. ALP avticity in CD73-/aortic root fragments incubated for 72h in osteogenic media, osteogenic media with ATP and osteogenic media with adenosine. All values are shown as mean ± SEM (n=4; Two-way ANOVA with post-hoc Tukey test and Student t test: *p<0.05; **p<0.01; ***p<0.001).

23

ACCEPTED MANUSCRIPT Figure 6. Decreased blood adenosine concentration and plasma total antioxidant status in CD73-/- mice. A) Blood adenosine concentration and B) plasma total antioxidant status of CD73-/- and WT mice. All values are shown as mean ± SEM, (n=21; Two-way ANOVA with

AC

CE P

TE

D

MA

NU

SC R

IP

T

post-hoc Tukey test and Student t test: *p<0.05; **p<0.01; ***p<0.001).

24

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

Figure 1

25

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

Figure 2

26

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

Figure 3

27

AC

CE P

TE

D

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

Figure 4

28

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

Figure 5

29

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

Figure 6

30

MA

NU

SC R

IP

T

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

Graphical abstract

31

ACCEPTED MANUSCRIPT Table 1. Protocols used for calcium deposits, elastic fibers and fibrin clots stainings.

Staining Technique

Staining Reagent

Component Stained

Red

Reddish brown

Red (Erythrocytes – yellow, Connective tissue – blue)

AC

CE P

TE

D

MA

NU

SC R

IP

1% aq Alizarin Red Calcium deposits in 10% ammonium hydroxide solution (pH 6.3 – 6.5) Orcein Stain 1% Orcein in 70% Elastic fibers alcohol solution with 1 % hydrochloric acid Martius Scarlet Blue Bouin’s fluid, Fibrin Stain Martius yellow, Crystal scarlet, Methyl blue

T

Alizarin Red Stain

Positive Colour

32

ACCEPTED MANUSCRIPT CONFLICT OF INTEREST

AC

CE P

TE

D

MA

NU

SC R

IP

T

None declared.

33

ACCEPTED MANUSCRIPT

HIGHLIGHTS  Deletion of CD73 leads to almost complete loss of adenosine production from AMP.

T

 CD73 knock-out causes increased peak aortic flow, aortic valve thickening and

SC R

CD73 activity is crucial for normal aortic valve function and preservation of its

CE P

TE

D

MA

NU

structure.

AC



IP

mineralization.

34