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Evaluation of a preventive therapy in a model of calcified aortic stenosis

Tableaux et figures

Introduction

Aortic stenosis (AS) is the most common form of valvular heart disease in developed countries [START_REF] Nkomo | Burden of valvular heart diseases: a population-based study[END_REF], affecting between 25% of patients over 65 years old and 40% over 80 years old [START_REF] Coffey | The Prevalence, Incidence, Progression, and Risks of Aortic Valve Sclerosis[END_REF][START_REF] Stewart | Clinical Factors Associated With Calcific Aortic Valve Disease[END_REF]. Given the lack of prevention strategies aimed at reducing the disease progression, surgical valve replacement was the unique therapeutic alternative until the recent development of revolutionary approach based on percutaneous transcatheter aortic valve implantation (TAVI) (4). However, not all the patients are candidates percutaneous intervention and despite numerous trials, no medical therapy demonstrated its ability to slow down the disease progression marked by high mortality in this case [START_REF] Marquis-Gravel | Medical Treatment of Aortic Stenosis[END_REF].

The early pathophysiological mechanisms involve mechanical lesions, chronic Inorganic pyrophosphate (PPi) is a metabolite naturally present in the human body and produced by the hepatic type 1 ectonucleotide phosphodiesterase enzyme (ENPP1) from adenosine tri-phosphate (ATP) supplied by the transporter ABCC6 [START_REF] Jansen | ABCC6mediated ATP secretion by the liver is the main source of the mineralization inhibitor inorganic pyrophosphate in the systemic circulation-brief report[END_REF].

Previous data emphasized the role of PPi in the inhibition of ectopic mineralization resulting from generalized arterial calcification of infancy with deficiency of ENPP1 or from pseudoxanthoma elasticum with ABCC6 mutation [START_REF] Jansen | ABCC6mediated ATP secretion by the liver is the main source of the mineralization inhibitor inorganic pyrophosphate in the systemic circulation-brief report[END_REF]. Several in vitro and in vivo studies have confirmed this role, including in cardiac and aortic tissues [START_REF] Rathan | The role of inorganic pyrophosphate in aortic valve calcification[END_REF][START_REF] O'neill | Treatment with pyrophosphate inhibits uremic vascular calcification[END_REF], and suggested that a direct binding of the PPi to the hydroxyapatite crystals could prevent their aggregation, selectively stopping the process of ectopic mineralization.

Based on these findings, the aim of this study was to assess the feasibility of inducing a calcified AS using targeted aortic valve irradiation in mice, and to evaluate the impact of a therapy using PPi to prevent aortic stenosis in this model.

Material and methods

Animals

The institutional animal ethics committee approved the animal experiments

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Three-dimensional anatomic atlas of aortic valve

To target the aortic valve during irradiation, we built a murine cardiac atlas using MR data. MR experiments were carried out using a 7T magnet (Pharmascan® Bruker, Billerica, USA). T1 Flash MR images were performed in 13 male C57Bl6/J mice 16week-old) using a strict axial multi-slice sequence encompassing the heart: TR/TE 176.6/4.2 ms, 20 slices, 6 repetitions. Automatic alignment and fusion of the 6 repetitions were performed using ImageJ software (version 1.52a, Bethesda, MD) to obtain one single image series for each animal. Aortic valve and aorta from the insertion to aortic isthmus were segmented. Then, a threshold set to 75% of colocalization of each aortic valve segmentation was performed to determine the aortic valve segmentation on the atlas. The same method was used for the aorta segmentation.

Irradiation protocol

Anesthesia was induced with 5% isoflurane gas (Forene®, AbbVie, Rungis, France) and maintained with isoflurane 2% gas in a mixed of O2 and N2O (1:2) and animals were placed in prone position in a dedicated bed. Computed tomography (CT) was performed encompassing the chest (80 kV, 0.5 mA). Using 3D Slicer software (www.slicer.org), cardiac atlas was manually aligned with CT acquisition to localize the aortic valve and the aorta with segmentations using trachea and bronchia bifurcation as anatomical markers. Planning of radiation exposure was performed using SmartPlan ® (Precision X-ray Inc., North Brandford, CT). Tissues (air, lung, soft tissue, bone) were segmented using Hounsfield units. Irradiation consisted of 2 beams of 2 mm diameter with an angle of 45° and 135° (Figure 1). The spatial resolution and dose distribution was previously assessed using polymethyl methacrylate (PMMA)

phantoms. The animals randomized to receive radiation therapy underwent a targeted irradiation on the aortic valve at a dose of 10 Gy using a Pxi225CX micro-irradiator (Precision X-ray Inc, North Brandford, CT) in a single fraction (2.22 Gy/min, 225 kV, 13 mA, 0.3 mm copper filter).

Echocardiography

Echocardiography was performed at baseline and 3-month follow-up in isoflurane anesthetized mice with a heart rate <450 beats/min using Philips iE33 MRI images were analysed using Osirix v.6.5.2 software. MPIO-αVCAM-1 binding results in a T2* signal void in the lumen of the vasculature.

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Histological analysis

AS mode validationl:

Mice were sacrificed 3 months after their inclusion in the study. Heart were dissected after the injection of 300 µL of KCl 1 M under a microscope, perfused with heparinized (50 U/mL) Phosphate Buffer Saline 5/100 (PBS) and cryomounted in optimal cutting embedding medium (CellPath, ThermoFisher Scientific, Waltham, USA). For each animal, eight 10-µm-thick slices were collected. Cryosections were placed in 5% silver nitrate solution for 30-60 min then fixed in 5% sodium-thiosulfate solution for 2-3 min. Sections were parallel to the valve plane and digitized using ScanScope CS (Leica Byosystems, Wetzlar, Germany). Tissue segmentation was manually performed using Aperio ImageScope software v12.3 (Leica Biosystem, Wetzlar, Germany). Aortic valve leaflet area, von Kossa area, the number of independent von Kossa structures and the tissue aortic sinus area were assessed.

Preventive effects of PPi:

Mice were sacrified 7 months after their inclusion in the study. The heart dissection technique was performed in identical conditions as in the AS model except that the heart was weighed and fixed in formalin. Finally, section of hearts was perpendicular to the valve plane. Sections were digitized and post processed as described in the AS model study. In addition to von Kossa staining to assess mineralization process, Alizarin red staining was used.

Statistical analysis

Values were expressed as mean ± SEM. A linear model analysis was used to evaluate the effect of the post-radiation time, the genotype and the radiation dose.

Post-hoc analysis was performed using Tukey HSD test. A logistic regression analysis was used to correlate the quantitative analysis of von Kossa staining and sinus lesion area with mean aortic gradient, aortic valve flow velocity and aortic wall compliance.

For proportions, the chi-square test was used to compare differences between groups.

Statistical analyses were performed using JMP 11 (SAS Institute, Cary, NC). A p value ≤ 0.05 was considered statistically significant.

Results

Radiation exposure was well tolerated in all animals. The animals' weight remained stable during all the study.

Echocardiography

AS model validation:

Table 1 summarizes echocardiographic findings. There was a left ventricle enlargement associated with an interventricular septum thinning and a decreased systolic function at 3 months follow-up. However, there was no impact of radiation on these functional parameters, except on the aortic root diameter that was lower in animals submitted to radiation exposure. Aortic root compliance at 3-month follow-up was significantly impaired after radiation therapy, related to a decrease in systolic aortic diameter (Table 2).

Preventive effects of PPi:

Table 3, summarizes echocardiographic findings. Over time there was a left ventricle enlargement associated with an interventricular septum systolic thinning, an increase in diastolic and systolic diameters. An increased interventricular septum thickness was found in TT mice at baseline after randomization, and throughout followup. Regarding the left ventricular ejection fraction (LVEF), a decrease was observed over time, from the 2th month of follow-up. Although there was no global effect of radiation exposure and PPi, in multivariate analysis, a LVEF decrease over time was only observed in NO TT / RT+ mice at 3-month (p= 0.044) and 5-month (p= 0.0058) follow-up vs. baseline. No significant difference was observed in TT / RT+ over time.

The systolic function also decreased in the NO TT/RT-subgroup at 5-month vs.

baseline (p= 0.0267). Regarding aortic root diameter and compliance, no effect of radiation exposure was observed (Table 4). The global effect from PPi on aortic compliance (p= 0.045) should be interpreted with caution. Indeed results were only significant at baseline and 1 st month of follow-up, then no difference were found between TT and NO TT.

Valve function assessment

As model validation:

The aortic flow velocity and the trans-aortic valvular gradients were significantly higher in ApoE -/-mice (p<0.0001, Table 5). However, radiation therapy resulted in a further increase of aortic flow velocity (p<0.001) and valvular gradients (p<0.01), especially in ApoE -/-mice. As shown in Figure 2, the impact of radiation on trans-aortic Doppler findings was dose-dependent.

Preventive effects of PPi:

A global effect of PPi was observed (Table 6), leading to a stability over the time in maximum (p< 0.0001; Figure 4) and mean flow velocity (p< 0.0001). The same results were observed for maximal (p< 0.0001) and mean gradients (p= 0.0002). The global effect of radiation was responsible for an increase in aortic maximum flow velocity (p= 0.0492) and maximal gradient (p= 0.0283) especially in NO TT / RT+ mice.

Indeed, in NO TT/RT+, there was a significant increase in maximal flow velocity at 3month (p= 0.0015) and 5-month follow-up (p= 0.0002) vs. baseline. However, no significant difference was found in TT/RT+ and TT/RT-mice over time. No aortic regurgitation was noted.

MR morphologic imaging findings

Preventive PPi resulted in a decreased left ventricular mass (Table 7), with a significant difference observed at 3-month follow-up between TT/RT+ and NO TT/RT+ (p= 0.0005) and between TT/ RT-vs. NO TT/ RT-(p= 0.0007). Regarding EF, ESV and EDV, no significant difference were found.

α-VCAM MPIO MR imaging findings

In RT-animals, α-VCAM MPIO MR imaging was normal in WT mice whereas 5/8 (60%) ApoE -/-demonstrated a T2* signal void (Table 8). The incidence of a T2* signal void was increased in RT+ animals. Logistic regression confirmed that radiation exposure was independently associated to a T2* signal void in the valve leaflets (p< 0.001), and that both genotype and radiation exposure were associated to a T2* signal void in the aortic sinus (p< 0.01 and p< 0.05 respectively).

Histological analysis

AS model validation:

Histological findings showed that, independently of radiation exposure, ApoE -/- mice had an increased aortic sinus lesion area compared to WT (Table 9). In addition, there was a significant remodeling of valve leaflets and aortic sinus lesion related to radiation exposure, especially in ApoE -/-mice. This remodeling further increased with the radiation dose in ApoE -/-. Von Kossa staining increased after radiation exposure in valve leaflets (p<0.001), especially in ApoE -/-mice. Of note, von Kossa staining was decreased in ApoE after 20 Gy compared to 10 Gy. As described in Table 10 and Figure 3, there was a significant correlation between tissue remodeling and both functional valve parameters and aortic wall compliance. Finally, radiation exposure resulted in left ventricular fibrosis assessed using Picro-Sirius red staining (p<0.05).

Preventive effects of PPi:

Histological assessment (table 11) showed a strong interaction between radiation exposure and PPi using von Kossa staining (p< 0.0001). Radiation exposure was responsible of an increase of 144% (mean) of von Kossa staining in aortic leaflets area in NO TT mice (p= 0.0007) (figure 5). In addition, a decrease of 52% (mean) of von Kossa staining was observed in TT/RT+ vs. NO TT/RT+ mice (p= 0.0057). Thus, no significant increase of Von Kossa staining was found in TT mice with radiation exposure. Surprisingly no significant difference was observed for Alizarin and Picro-Sirius red stainings (in leaflet area), leaflet area and sinus tissue area.

Discussion

The main result of this study is that radiation exposure of ApoE -/-mice resulted in an aortic stenosis demonstrated by an increase of aortic flow velocity associated with an increase of von Kossa staining in aortic valve leaflets. In addition, early treatment with PPi prevented the appearance of radiation-induced aortic lesions.

Calcified AS is characterized by fibro-calcific remodeling of valve leaflets. The progression of the disease involves severe calcification within the valve leaflets leading to an impairment of valve motion contributing to a blood flow obstruction [START_REF] Lindman | Calcific aortic stenosis[END_REF]. The high incidence of valvular dysfunction had been reported in populations undergoing mediastinal radiation therapy [START_REF] Wethal | Valvular dysfunction and left ventricular changes in Hodgkin's lymphoma survivors. A longitudinal study[END_REF]. The progression of AS is also accelerated in these patients, underlining the potential of X-rays to initiate and develop valvular lesions [START_REF] Donnellan | Rate of Progression of Aortic Stenosis and its Impact on Outcomes in Patients With Radiation-Associated Cardiac Disease[END_REF].

Previous in vitro studies demonstrated that a local 10 Gy irradiation of human aortic valve interstitial cells induced an osteogenic phenotype differentiation (8).To our best knowledge, the present study is the first that evaluated the impact of targeted radiation exposure on the development of a calcified AS in a mouse model.

Calcific deposition has been previously reported in a mouse model of high fat diet-induced AS [START_REF] Aikawa | Multimodality Molecular Imaging Identifies Proteolytic and Osteogenic Activities in Early Aortic Valve Disease[END_REF][START_REF] Scatena | Increased Calcific Aortic Valve Disease in response to a diabetogenic, procalcific diet in the LDLr -/-ApoB 100/100 mouse model[END_REF]. The supplementation of 1.25% of cholesterol, 57.5% kcal fat and 27.4% kcal carbohydrate resulted in valve leaflets thickening with an increase in the sinus lesion volume compared to control mice fed with a normal diet [START_REF] Scatena | Increased Calcific Aortic Valve Disease in response to a diabetogenic, procalcific diet in the LDLr -/-ApoB 100/100 mouse model[END_REF]. A valve leaflet thickening was also found in Ldlr -/-Apob 100/100 mice with a 0.15 % cholesterol diet, associated with an increase of aortic valve flow velocity which was rescued by a regular exercise training [START_REF] Matsumoto | Regular exercise training prevents aortic valve disease in low-density lipoprotein-receptordeficient mice[END_REF]. In our study, although leaflet thickness tended to increase with the radiation dose, increasing radiation from 10 to 20 Gy in ApoE -/-mice resulted in lower leaflet calcification von Kossa staining. A similar phenomenon was observed with the amount of aortic sinus lesion that further increased with radiation dose, suggesting that active inflammation is responsible for persistent increasing leaflet thickness.

Left ventricular remodeling is a key issue in patients with aortic stenosis [START_REF] Lindman | The adverse impact of diabetes mellitus on left ventricular remodeling and function in patients with severe aortic stenosis[END_REF].

Using various preclinical models, previous studies showed an association between aortic valve leaflet remodeling and left ventricular function impairment. A significant decrease of fractional shortening was found in several models [START_REF] Barrick | Reduced EGFR causes abnormal valvular differentiation leading to calcific aortic stenosis and left ventricular hypertrophy in C57BL/6J but not 129S1/SvImJ mice[END_REF][START_REF] Nus | Diet-Induced Aortic Valve Disease in Mice Haploinsufficient for the Notch Pathway Effector RBPJK/CSL[END_REF], as well as a left ventricular hypertrophy [START_REF] Barrick | Reduced EGFR causes abnormal valvular differentiation leading to calcific aortic stenosis and left ventricular hypertrophy in C57BL/6J but not 129S1/SvImJ mice[END_REF][START_REF] Drolet | A high fat/high carbohydrate diet induces aortic valve disease in C57BL/6J mice[END_REF][START_REF] Weiss | Calcific aortic valve stenosis in old hypercholesterolemic mice[END_REF][START_REF] Miller | Evidence for active regulation of pro-osteogenic signaling in advanced aortic valve disease[END_REF]. In our study, Picro-Sirius red staining showed an increased left ventricular fibrosis after radiation exposure. Moreover, based on MRI findings, it confirmed the presence of LV hypertrophy after radiation exposure. Barrick et al. [START_REF] Barrick | Reduced EGFR causes abnormal valvular differentiation leading to calcific aortic stenosis and left ventricular hypertrophy in C57BL/6J but not 129S1/SvImJ mice[END_REF] reported LV hypertrophy and dilation with a decreased FS in Egfr wa2/wa2 mice with a higher peak aortic velocity of 370 cm/s. Compared to our results, studies based on high cholesterol diet reported LV hypertrophy in spite of similar aortic velocities [START_REF] Barrick | Reduced EGFR causes abnormal valvular differentiation leading to calcific aortic stenosis and left ventricular hypertrophy in C57BL/6J but not 129S1/SvImJ mice[END_REF][START_REF] Drolet | A high fat/high carbohydrate diet induces aortic valve disease in C57BL/6J mice[END_REF][START_REF] Weiss | Calcific aortic valve stenosis in old hypercholesterolemic mice[END_REF].

The second main result from our study concerns the impact of PPi to prevent AS. First, based on echocardiography findings, PPi prevented from increase of transvalvular aortic velocity while no significant LVEF decrease was found in TT mice in contrary to NO TT mice. Moreover, based on MRI findings, PPi prevented LV hypertrophy. Finally, histological findings showed that PPi prevented the increase of von Kossa staining on aortic leaflets after radiation exposure.

Histological findings highlighted discrepancies in the results concerning the mineralization process using von Kossa staining. First, a lower quantification of von Kossa staining in aortic leaflets from 10 to 20 Gy which may suggests a toxic effect of high dose radiation on the mineralization process, with persistent inflammation as demonstrated by MPIO-αVCAM1 MR imaging. Second a discrepancy between von Kossa and Alizarin red staining should be interpreted with caution. It is documented that melanocytes, from neural crest cells, are consistently found in heart valves, including in aortic leaflets [START_REF] Levin | Melanocyte-like cells in the heart and pulmonary veins contribute to atrial arrhythmia triggers[END_REF], and can be misidentified as calcification using von Kossa staining [START_REF] Hulin | Maturation of heart valve cell populations during postnatal remodeling[END_REF]. However, the cardiac function of melanocytes remains poorly understood. The increase of von Kossa staining in NO TT / RT+ was related to radiation exposure. Radiation may induce DNA damage which is responsible for an increase of alpha melanin stimulating hormone (α-MSH), an agonist of the melanocortin 1 receptor (MC1R) [START_REF] Lin | Melanocyte biology and skin pigmentation[END_REF][START_REF] Suzuki | Radiation-induced DNA damage and delayed induced genomic instability[END_REF]. Activation of MC1R in melanocytes result in melanin secretion.

However, the rationale for the absence of increase in TT/RT+ is less clear.

The absence of Alizarin red staining suggest that the observed von Kossa staining variations may be linked to melanin secretion and supports imaging findings as well. Previous findings demonstrated that melanin may affect the mechanical properties of heart valves [START_REF] Carneiro | Relationships between melanocytes, mechanical properties and extracellular matrix composition in mouse heart valves[END_REF][START_REF] Balani | Melanocyte pigmentation stiffens murine cardiac tricuspid valve leaflet[END_REF]. The amount of melanin is strongly and positively correlated with valvular rigidity in mice. In that way, based on our histological findings, increase of von Kossa staining linked to melanin could participate to the increase of transvalvular aortic velocity flow. Melanin is colocalized with a proteoglycan from the spongiosa heart valve matrix, the versican B [START_REF] Carneiro | Relationships between melanocytes, mechanical properties and extracellular matrix composition in mouse heart valves[END_REF]. The rigidity could be due to melanin directly or due to an interaction with the extra cellular matrix as Versican B.

Melanocytes produce metalloproteases and could be involved in tissue homeostatis in interaction with extra cellular matrix as Versican B. This proteglycan play a key role in the neural crest migration as melanoblasts [START_REF] Perissinotto | Avian neural crest cell migration is diversely regulated by the two major hyaluronanbinding proteoglycans PG-M/versican and aggrecan[END_REF][START_REF] Braunewell | Functional involvement of sciatic nerve-derived versican-and decorin-like molecules and other chondroitin sulphate proteoglycans in ECM-mediated cell adhesion and neurite outgrowth[END_REF].

The absence of increase von Kossa staining in TT/RT+ is less clear. We hypothesize that PPi allows to create a protective and less critical environment without the need for melanocyte action. Indeed many role of cardiac melanocytes are known in cardio-vascular field. MC1R is also present in endothelial cells, monocytes and fibroblasts [START_REF] Böhm | The role of melanocortins in skin homeostasis[END_REF][START_REF] Catania | Targeting melanocortin receptors as a novel strategy to control inflammation[END_REF]. These cells play a key role in the regulation of melanogenesis and interact between each other in the skin [START_REF] Yamaguchi | Physiological factors that regulate skin pigmentation[END_REF]. MC1R is activated by a-MSH which is secreted by melanocytes. The MC1R and a-MSH signaling pathway has a protective role on human aortic endothelial function [START_REF] Rinne | α-Melanocyte-stimulating hormone regulates vascular NO availability and protects against endothelial dysfunction[END_REF][START_REF] Saporiti | Melanocortin-1 Receptor Positively Regulates Human Artery Endothelial Cell Migration[END_REF], inflammation [START_REF] Catania | Targeting melanocortin receptors as a novel strategy to control inflammation[END_REF] and atherosclerotic processes [START_REF] Rinne | Pharmacological activation of the melanocortin system limits plaque inflammation and ameliorates vascular dysfunction in atherosclerotic mice[END_REF]. It activates a cholesterol efflux transporter in macrophages, reduces oxidized LDL uptake and allows to stabilize atherosclerotic plaques [START_REF] Rinne | Melanocortin 1 Receptor Signaling Regulates Cholesterol Transport in Macrophages[END_REF]. Concerning mineralization process, cell culture showed that a-MSH may also induce osteoblastic differentiation of MC3T3 cells with an increase of ALP expression and RUNX2 activation [START_REF] Wang | α-melanocyte stimulating hormone (α-MSH) promotes osteoblast differentiation of MC3T3-E1 cells[END_REF]. Based on these arguments, we hypothesize that melanocytes could play a role in the development of aortic valve lesions after X-ray exposure.

Conclusion

This study demonstrated that targeted aortic valve irradiation in ApoE-/-mice resulted in aortic lesions with accelerated transvalvular flow. Early oral administration of PPi prevented the radiation-induced aortic stenosis, and confirm the usefulness of this preclinical model for assessing new therapeutic strategies. Understanding the role of melanocytes in aortic leaflets warrant further investigations.

  DOCTEUR EN MÉDECINE Présentée et soutenue publiquement le : 14 septembre 2020 par Mr Kevin PRIGENT Né le 22 avril 1991 à Rennes (35) : Évaluation d'une thérapeutique préventive chez un modèle de rétrécissement aortique calcifié. Président : Monsieur le Professeur Denis AGOSTINI Membres : Monsieur le Professeur Ludovic BERGER Monsieur le Professeur Farzin ESMAIL-BEYGUI Monsieur le Docteur Damien LEGALLOIS Directeur de thèse : Pr Alain MANRIQUE présidence de cette thèse. Je le remercie également pour son enseignement tout au long de mon internat. Veuillez recevoir l'expression de ma sincère reconnaissance. Messieurs les Professeurs Ludovic Berger et Farzin Esmail-Beygui ainsi que Monsieur le Docteur Damien Legallois, pour m'avoir fait l'honneur d'accepter de faire partie du jury et pour l'intérêt que vous avez porté à ce sujet de thèse. Mon directeur de thèse, le Pr Alain Manrique sans qui ce travail n'aurait jamais été possible. C'est un plaisir de travailler ensemble. Je suis reconnaissant pour ses conseils avisés, son expérience et sa confiance envers moi pour accomplir ce travail. Toute l'équipe de l'EA4650 du centre Cycéron dans laquelle j'ai pu évoluer dans le cadre de ce travail. Je remercie en particulier Céline Brocquehaye pour son accueil chaleureux et Guillaume Rucher pour notre collaboration. Je remercie Quentin Dupas pour son aide et son humour. Je remercie également chaleureusement le Docteur Christophe Simard pour son soutien, sa disponibilité & son écoute. L'équipe du CMABio3, Maëlle Guyot et Nicolas Elie pour leurs expertises en Histologie. Adèle Riot pour ton aide précieuse sur la lecture des lames d'histologie qui a aidé à la mise au point de la technique. Le Professeur Philippe Delaval, pneumologue et ancien Doyen de la faculté de Médecine de Rennes, qui a incarné pour moi une figure dans mes études supérieures. Je garderai le souvenir de sa sagesse aussi grande que son humilité. Au Docteur Tanguy Rohou, radiologue, qui m'a fait découvrir et aimer l'Imagerie Médicale par son travail rigoureux et son partage spontané de ses connaissances. Mes parents pour leurs soutiens et leurs encouragements depuis toujours et durant ces longues années d'études. Mon frère Gwénolé et ma soeur Aurélie. Merci à tous les deux pour vos conseils, votre écoute et votre soutien. Je suis fier de vos brillants parcours à tous les deux et de ce que vous êtes aujourd'hui. Tous mes amis, notamment ceux de mon lycée René Cassin, de la faculté de Médecine de Rennes 1 et mes co-internes du C.H.U de Caen qui ont été présents de près comme de loin et qui m'ont permis d'appréhender ce long parcours de manière plus sereine. Adèle, pour tout ce que tu es. Merci pour ta patience, ton soutien et ton aide si précieuse depuis toutes ces années. A mon tour de t'accompagner pour la fin de ton parcours en Médecine et bien au-delà.

  MPIO: Micron-sized particles of iron oxide MR: Magnetic Resonance NO TT: Group without treatment NO TT/RT+: Group without treatment but radiation exposure NO TT/RT-: Group without treatment and without radiation exposure PBS: Phosphate Buffer Saline PMMA: Polymethyl methacrylate PPi: Pyrophosphate inorganic RA: Rétrécissement aortique RUNX2: RUNt-related transcription factor 2 SEM: Standard error of the mean TAVI: Transcatheter aortic valve implantation TT: Group with treatment TT/RT+: Group with treatment and with radiation exposure TT/RT-: Group with treatement and without radiation exposure VCAM: Vascular Cell Adhesion Molecule VIC: Valve interstitial cells WT: Wild Type

Figure 1 .Figure 2 .

 12 Figure 1. Planning of radiation exposure. Irradiation consisted of 2 X-ray beams of 2 mm diameter with an angle of 45° and 135°.

Figure 3 .

 3 Figure 3. Correlation between von Kossa area and transaortic flow velocity, mean gradient and aortic wall compliance.

Figure 4 .

 4 Figure 4. Impacts of radiation and PPi on evolution of velocity flow using echocardiography. Analysis found a global effect (p<

Figure 5 .

 5 Figure 5. Impacts of radiation and PPi on aortic leaflets using von Kossa staining histological assessment. Analysis found a

  inflammation, and osteogenic phenotypic changes of valve interstitial cells inducing progressive mineralization[START_REF] Lindman | Calcific aortic stenosis[END_REF] that is central to the development of this disease. The development of new preventive or therapeutic strategies suffers from the lack of reliable and reproducible preclinical models of calcified aortic stenosis. Documented preclinical models are mainly based on accelerated atherosclerosis in Ldlr -/-and ApoE - /-mice submitted to high fat diet. Clinical studies demonstrated that mediastinal radiation therapy may induce various cardiac damage including calcified AS (7). In addition, recent in vitro experiments demonstrated that irradiation of human aortic valve interstitial cells (VIC) induced the expression of osteogenic factors 24h after irradiation such as Bone Morphogenetic Protein 2 (BMP-2), alkaline phosphatase (ALP) and Runx2 that are necessary for bone formation[START_REF] Nadlonek | Radiation Induces Osteogenesis in Human Aortic Valve Interstitial Cells[END_REF].

  Philips Healthcare, Best, Netherlands) and linear ultrasound probe L15-7io (128 elements, 7-15 MHz). Aortic valve function was assessed using pulse wave Dopplermode that measures transaortic flow velocity and mean gradient. M-mode images of the parasternal long and short axis views were used to measure left ventricle and aortic root dimensions. In addition, 16 animals randomized to PPi treatment or placebo underwent addition echocardiography assessment at 2 months and 5-month follow-up. The mean of 3 measurements for each parameter was calculated. Cardiac MR imaging Morphogical evaluation in cardiac MR only concerned the effects of preventive PPi. Eight animals from each subgroup (n = 32) had a follow-up imaging. MRI was performed using a 7T magnet (Pharmascan® Bruker, Billerica, USA) with dual respiratory and ECG gating with a heart rate < 500 beats/min. It was performed at baseline then 1 st , 3 rd (n = 32) and 5 th month (n = 16) from radiation exposure or PPi introduction. A multi-slice black blood intragate flash sequence was used: thickness of the sections of 560 μm, field of view: 28 × 28 mm, size of the matrix: 128 × 128 , spatial resolution: 0.219x0.219 mm / pixel, 20 images / cardiac cycle, TE / TR: 2/73, tilt angle: 25 °. According to the animal and its growth, the number of slices were adapted to the heat size and could varied from 8 to 11. The post image processing was done using the software Segment © version 2.0. A manual segmentation of the two ventricles contours were done at the endocardial and epicardial level in telesystole and telediastole in order to calculate the left ventricular volumes, ejection fraction and mass. Left ventricular mass and volumes were indexed to the animal body weight measured just before each examination. To evaluate endothelial activation, MR imaging without and with intravenous injection of 200 μL of micron-sized particles of iron oxide (MPIO) (DynaBeads MyOnes Tosyl Activated, ThermoFisher Scientific, Waltham, USA) conjugated with specitic αVCAM-1 antibodies (clone A(429), BD BioScience, Franklin Lakes, USA) were used by incubation at 37°C for 48h. Telediastolic T2* weighted MR images were acquired using a multi-slice sequence: field of view: 1809 x 939 mm2, TR/TE 100/4.25 ms, 12 slices, voxel: 0.1x0.1x0.15 mm3 encompassing the thoracic aorta were performed were performed in 2 WT RT-, 4 WT 10 Gy, in 3 WT 20 Gy, in 4 ApoE -/-RT-, in 4 ApoE -/-10 Gy and in 6 ApoE -/-20 Gy mice.

Table 1 . Left ventricle, left atrium and aorta dimensions assessed using echocardiography at baseline and 3-month follow- up.

 1 Measurements were performed in 22 WT and 25 ApoE -/-mice. IVS.: interventricular septum, LVD: left ventricle diameter, LVPW:

	left ventricle posterior wall, EF: ejection fraction, FS: fractional shortening, LA: left atrium diameter, d: diastole, s: systole. Data are
	expressed as mean ± SEM.
		Baseline		3 months		p-value
		WT	ApoE -/-	WT	ApoE -/-	Global	Time	Genotype	Radiation
	IVS.d (mm)	0.72±0.02	0.75±0.01	0.67±0.02	0.68±0.01	0.0009	0.0002	ns	ns
	IVS.s (mm)	0.89±0.05	0.8±0.02	0.72±0.02	0.76±0.03	0.01	0.0005	ns	ns
	LVDd (mm)	3.70±0.06	3.75±0.09	3.95±0.06	4.12±0.09	<0.01	0.0002	ns	ns
	LVDs (mm)	2.48±0.07	2.59±0.08	2.84±0.06	3.13±0.1	<0.0001	<0.0001	0.01	ns
	LVPWd (mm)	0.75±0.02	0.86±0.04	0.82±0.03	0.83±0.03	ns	-	-	-
	LVPWs (mm)	0.98±0.03	1.07±0.04	0.98±0.03	0.98±0.04	ns	-	-	-
	FS (%)	33.13±1.32	31.15±1.08	28.27±0.67	24.40±1.23	<0.0001	<0.0001	<0.01	ns
	EF (%)	68.42±1.7	65.30±1.55	61.30±1.08	54.41±2.00	<0.0001	<0.0001	<0.01	ns
	LA (mm)	2.17±0.05	2.36±0.07	2.80±0.08	3.15±0.07	<0.0001	<0.0001	0.0003	ns
	Aorta (mm)	1.79±0.02	1.86±0.03	1.88±0.04	1.81±0.03	<0.01	ns	ns	0.0001

Table 2 . Systolic, diastolic and aortic wall compliance measurements assessed using echocardiography. WT

 2

	control (n=10),

Table 3 Effects of PPi administration on left ventricle and aortic dimensions assessed using echocardiography at baseline, 3 months and 5 months of follow

 3

		Baseline		3 months		5 months		p-value
		NO TT	TT	NO TT	TT	NO TT	TT	Global	Time	PPi	Radiation
		n=16	n=16	n=16	n=15	n=7	n=8
	IVSd (mm) 0.81+0.028 0.86+0.037 0.89+0.029 0.84+0.042 0.97+0.064	0.91+0.031	0.0237	0.0844 0.0470	ns
	IVSs (mm) 0.97+0.036 1.20+0.035	1.2+0.034	1.3+0.058	1.3+0.11	1.4+0.076	< 0.001 < 0.001 0.0006	ns
	LVDd (mm) 3.31+0.09	3.37+0.062	3.6+0.062	3.5+0.13	3.9+0.074	3.9+0.095	ns	0.0012	ns	ns
	LVDs (mm) 1.91+0.12	2.05+0.057	2.4+0.061	2.2+0.14	2.7+0.14	2.6+0.14	< 0.068	0.0007	ns	ns
	LVPWd	0.88+0.035 0.99+0.059 0.97+0.061	1.1+0.10	0.89+0.036	0.90+0.033	ns	ns	0.0037	ns
	(mm)
	LVPWs	1.23+0.056 1.29+0.059	1.3+0.053	1.3+0.11	1.2+0.043	1.2+0.057	ns	ns	0.0467	ns
	(mm)
	FS (%)	42.99+2.44 39.03+1.51	35+1.1	37+1.9	31+3.0	34+2.3	0.0677	0.0025	ns	ns
	EF (%)	79.69+2.2	75.49+1.9	71+1.4	73+2.3	65+4.3	70+2.9	ns	0.0027	ns	ns
	Aorta (mm) 1.708+0.035 1.652+0.034 1.823+0.041 1.739+0.043 1.824+0.082 1.755+0.037	0.0317	0.006	0.005	ns

-up. N: mice number, IVS: interventricular septum, LVD: left ventricle diameter, LVPW: left ventricle posterior wall, EF: ejection fraction, FS: fractional shortening, LA: left atrium diameter, d: diastole, s: systole. Data are expressed in mean ± SEM.

Table 4 . Effect of preventive PPi on the aortic root assessed using echocardiography. Aortic

 4

	wall compliance was calculated

Table 5 . Functional aortic valve assessment using echocardiography. Data

 5

	are expressed as mean±SEM, *p<0.05 vs. ApoE-/-

Table 6 . Functional aortic valve assessment at 3 months according to preventive PPi treatment. Data

 6

	are expressed in

Table 7 . Preventive effect of PPi administration on MRI results. Data

 7 are expressed as mean±SEM and are indexed to the weight

	of the animal. LVM: left ventricle mass, EDV: end diastolic volume, ESV: end systolic volume, SV: stroke volume, EF: ejection fraction.
		NO TT		TT		p-value
		RT-	RT±	RT-	RT±
						Global	Time	PPi	Radiation
		(n=8)	(n=8)	(n=7)	(n=8)
	LVM (mg/g)	3.434±0.14	3.7±0.12	2.6±0.91	2.8±0.13	<0.0001 0.0141	0.0013 ns
	EDV (µl /g)	2.437±0.16	2.451±0.15	2.8±0.079	2.80±0.2	0.0054	0.0041	0.0658 ns
	ESV (µl /g)	1.08±0.1	1.225±0.125	1.5±0.093	1.7±0.19	0.0006	0.0002	0.047	ns
	SV (µl /g)	0.0013±0.0001 0.0013±0.0001 0.0013±0.0001 0.0013±7.5 e -005 ns	ns	ns	ns
	EF (%)	55.78±2.18	55.78±2.18	47±3.4	48±3.7	0.0002	<0.0001 0.0838 0.0328

Table 8 . Proportion of MPIO -αVCAM-1 binding using MR imaging in aortic valve leaflets and aortic sinus. MPIO

 8

	-αVCAM-1

Table 9 .

 9 Histologic assessment. Fibrosis was assessed using Picro-Sirius red staining and mineralization process using von Kossa staining. Fibrosis corresponds to the ratio between Picro-Sirius red area and left ventricular area and von Kossa area to the % of von Kossa staining in the aortic valve leaflets area. All the parameters were assessed in 4 WT RT-, 3 WT 10 Gy, 3 WT 20 Gy, 4 ApoE -/- RT-, 4 ApoE -/-10 Gy and 3 ApoE -/-20 Gy. Data are expressed as mean ± SEM, *p<0.05 vs. ApoE RT- † p<0.05 vs. ApoE 10 Gy § vs.

	dose-equivalent WT.
		RT-	10	20	RT-	10	20	Global	Genotype Radiation
	Fibrosis (%)	10.25±0.01	8.83±0.02	11.72±0.01	5.89±0.01	14.30±0.02	9.68±0.02	<0.05	ns	<0.05
	Leaflet area
		0.144±0.006 0.161±0.015 0.166±0.015 0.115±0.005 0.164±0.008*	0.195±0.019*	0.0001	ns	0.0001
	(mm²)
	Sinus tissue	0.253±0.010 0.245±0.009 0.217±0.022 0.473±0.030 § 0.484±0.026 §	0.697±0.034* † §	<0.0001 <0.0001	<0.05
	area (mm²)
	Von Kossa	4.187 ±11.7 3.809±4.4	5.224±1.62	4.224±6.76	7.027±10.287* 4.027±4.584* †	<0.0001 ns	<0.0001
	Area (%)

Table 10 . Correlation between aortic valve function using echocardiography and histological analysis

 10

		Flow velocity		Mean gradient		Aortic wall compliance
		r	p	r	p	r	p
	Mineralization	0.57	<0.01	0.55	0.02	0.47	<0.05
	Leaflet area	0.65	0.002	0.62	<0.001	0.48	<0.05
	Aortic sinus lesion 0.55	0.02	0.60	<0.01	ns	ns

. p and r values of logistic regression analysis between transaortic flow velocity, mean gradient and aortic wall compliance with von Kossa area (mineralization), leaflet area and aortic sinus lesion.

Table 11 .

 11 Histologic assessment. Fibrosis was assessed using Picro-Sirius red staining and mineralization process using von Kossa staining. Fibrosis correspond to the % of red staining in the leaflet tissue area. Von Kossa area correspond to the % of von Kossa in the aortic leaflet area. Alizarin red area correspond to the % of red staining in the aortic leaflet area Data are expressed in mean ±

	SEM.
			NO TT		TT	p-value
		RT-	RT+	RT-	RT+	Global	PPi	Radiation
	Fibrosis (%)	13.55±0.968	12.19±0.956	13.75±1.532	12.88±0.673	ns	ns	ns
	Leaflet area
	(mm 2 )	0.157±0.02	0.157±0.02	0.130±0.013	0.174±0.021	ns	ns	ns
	Sinus tissue
	area (mm 2 )	1.096±0.142	1.132±0.114	1.048±0.1	0.969±0.111	ns	ns	ns
	Von Kossa
		0.977±0.1808	2.384±0.2465	1.930±0.3413	1.135±0.2166	0.0003	ns	ns
	Area (%)
	Alizarin Red
		0.0721±0.025	0.0979±0.045	0.0459±0.016	0.2112±0.117	ns	ns	ns
	Area (%)

Conclusion_____________________________________________________ 16 Funding________________________________________________________ 17 Préambule

  Ce choix s'est basé sur la littérature, montrant le développement de calcifications ectopiques au sein des tissus mous et notamment vasculaires dans des pathologies caractérisées par un déficit en PPi. Cela suggérait un rôle majeur du PPi sur la minéralisation. Ce rôle s'est confirmé par des études in vitro et in vivo sur des lésions aortiques et myocardiques. Le second objectif était également de démontrer la

	Le rétrécissement aortique (RA) est la valvulopathie la plus fréquente. Son
	évolution est inéluctable avec un pronostic sombre en absence de traitement. Le
	remplacement prothétique valvulaire est l'unique option efficace. Pour les patients non
	candidats à ce type de prise en charge, l'absence de traitement médical efficient en
	font actuellement un problème de santé publique majeur. Le développement de
	nouveaux agents est primordial et passe par l'évaluation préclinique chez l'animal. Or,
	l'évaluation de nouvelles stratégies thérapeutiques souffre d'un manque de modèles
	précliniques fiables et reproductibles du RA. Il apparaît donc nécessaire de développer
	un nouveau modèle pour évaluer de nouveaux agents thérapeutiques.

Mon projet dans le cadre de ma thèse d'exercice en Médecine s'est inscrit dans la suite du développement d'un modèle préclinique de RA au sein de l'EA4650 par Mr Guillaume Rucher & le Pr Alain Manrique. Ce modèle s'est basé sur des études ayant démontré une association entre le RA et la radiothérapie. Pour induire le RA, une irradiation ciblée de la valve aortique a été effectuée chez des souris transgéniques ApoE -/-présentant une athérosclérose accélérée. Les résultats obtenus étaient en faveur d'un RA.

Mon objectif principal était d'évaluer l'efficacité d'une thérapeutique préventive chez ce modèle. À ce titre, j'ai rédigé la saisine du projet #18217 dans une étude comportant 60 souris ApoE -/-réparties en groupe contrôle vs. traité. J'ai eu recours à l'irradiation ciblée de la valve aortique chez la souris ApoE -/-(10Gy) afin de sensibiliser les mécanismes de minéralisation. Afin de tenter de ralentir l'évolution du RA, le pyrophosphate inorganique (PPi) a été choisi comme agent thérapeutique potentiel. reproductibilité du modèle radique de RA pour venir conforter les résultats préliminaires.

Remerciements

Je tiens à remercier très sincèrement, Monsieur le Pr Denis Agostini, pour m'avoir fait l'honneur d'accepter la

Funding :

This work was conducted as part of the FHU REMOD-VHF project and supported by the French Government, managed by the National Research Agency (ANR) under the program "Investissements d'avenir" with the reference ANR-16-RHUS-0003.

RESUME DE LA THESE EN ANGLAIS :

Introduction: Mediastinal radiotherapy is a major cause of cardiac complications including aortic stenosis (AS). However, there is a lack of a preclinical model of AS mimicking radiation-induced lesions. In addition, AS is the most frequent valvular disease. In inoperable patients without effective medical treatment, the prognosis is poor. This project consists in evaluating by Imaging and Histology, the effectiveness of a preventive therapy in a mouse model of AS. Materials and methods: Targeted irradiation of the aortic valve (10 or 20Gy) in C57 / Bl6 / J (WT) and ApoE -/ -mice was carried out to develop an AS model. Once the model had been developed, inorganic pyrophosphate (PPi) was studied as a preventive therapy in ApoE -/ -(0 or 10Gy) mice. The assessment tools were echocardiography and cardiac magnetic resonance imaging. Cardiac and valve remodeling was studied in Histology using von Kossa and Rouge Picro-Sirius stains.