Jérôme Crépin

Thierry Bretheau

Daniel Caldemaison

J Crepin

Cavity growth and rupture of β-treated zirconium: A crystallographic model

de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Zirconium, a material mainly used in the nuclear industry, is characterized by the existence of two allotropic varieties, the f3 phase and the c,; phase which are b.c.c. and h.c.p. crystals, respectively. The former is found above, and the later below 1138 K. The grade 702 zirconium tested had undergone an c,;--+ f3--+ c,; cycle at a slow cooling rate ( -15° /s) inducing a bainitic transformation. The resulting microstructure consists of colonies of parallel laths with the same crystallographic orientation; the "lath boundaries" are planes where the precipitates of betagenic elements (Fe, Cr, Ni) locate. Three deformation mechanisms are activated, prismatic slip (1010)(1210), (10I2)<10TI) twinning and (1121)(1126) twinning, depending on the colony crystallographic orientation with respect to the tensile axis in accordance with Schmid's law. Prismatic slip is either homogeneous or concentrated in coarse slip lines along the lath boundaries [l]. This last mechanism is the most frequent one.

CHARACTERIZATION OF DAMAGE AND RUPTURE

Experimental procedure

Flat tensile test specimens (20 x 5 x 2 mm) were polished mechanically and electrolytically (U = 22 V, T = 300 K, t = 30 s, in CH3COOH: 80%, HCL04:20%). Then they were chemically etched (HF:22%, HN03: 11 %, glycerol:67%, T = 300 K, t � 5 s) to reveal the microstructure.

To follow on the sample surface the deformation mechanisms and the growth rate of damage, a Raith tensile stage located inside a SEM was used with a strain rate of the order of 10-4 s-1 • Fiducial 1 mm 2 gold grids with 5 µm pitch deposited on the surface of the sample permit one to follow qualitatively and quantitatively the local deformation mechanisms [2].

The local crystallographic orientation of crystal domains a few µm 3 large is determined with Electron Backscattering Diffraction (EBSD). This technique, which involves indexing the Kikuchi lines, provides the orientation of the studied crystal relative to a macroscopic reference axis attached to the sample tensile axis. Consequently, with the approximation of uniaxial stress, the Schmid factor of any deformation mechanism can be calculated in each grain.

The sample surfaces during a tensile test

At the surface of tensile test samples, damage cavities are observed for a global strain between 3 and 6%, far before any necking. Ductile rupture occurs at a global strain of about 10%, necking remaining weak in any case. The first damage cavities always appear in colonies subjected to prismatic slip either concentrated or homogeneous, but never in colonies subjected to twinning; they appear on the sample surface between the precipitates located at the lath boundaries or inside the lath between the last coarse slip line and the remaining undeformed part of the lath. In a colony, the cavities form alignments parallel to the lath boundaries (Fig. 1 ). During this growth, the cavities remain contained within one lath and their ultimate size is of the order of the thickness of the lath which is 10 µm. Cavities can show a peculiar hexagonal shape. Cavity coalescence occurs inside the alignments (Fig. 2) with the possibility of rupture of the ligament between two neighboring alignments. The absence of deformation exhibited by fi ducial grids for the laths which are not affected by voids shows that damage remains confined along the lath boundary (Fig. 1 ). Another type of damage has been observed at the inter-colony boundaries; it is due to the strong strain incompatibility between colonies which stemmed from a same former f3 grain and have a misorienta tion of either 60 or 90° (this misorientation is a consequence of the allotropic transformation). This damage development involves the activation of prismatic slip or twinning according to the orien tation of the grain with respect to the tensile axis [l]. Rupture occurs along the cavity alignments. The connection of the cracks developed inside adjacent colonies proceeds either by rupture of the damaged intercolony segregation zones or of the lath extremities.

The rupture surface

In Fig. 3 the rupture does not seem to be ductile because small circular dimples on the rupture surface are absent. It shows large areas of parallel elongated shapes with a long axis of several hundred micrometers and a small axis of about 10 µm corresponding to the size of the colonies. The comparison of the two associated rupture surfaces allows one to conclude that these elongated shapes are large elongated dimples. Such dimples called "fl uting zones" were described by Aitchinson and Cox [3] and Spurrier and Scully [4] in the case of stress-corrosion rupture. They reported that the long axis of these dimples is parallel to the local crystalline ( c > axis. Some zones constituted of small rounded dimples are also detected in the rupture surface, but found to be less numerous than the elongated ones. EDS analysis shows that the zones with small rounded dimples are rich in intermetallic precipitates, whereas zones with elongated large dimples do not have such precipitates.

Relation between the two observed surfaces

The necessary coherence between the different observations, the sample surfaces and the rupture surface, allows one to characterize the cavity morphology. If the same cavity is observed either on the sample surface [Fig. 4(a)] or on the rupture surface [Fig. 4(b)] it shows, without ambiguity, that it is tubular; an EBSD analysis of the lath orientation permits one to conclude that the tube axis is parallel to the (c > crystalline axis. Some of the cavities have a hexagonal cross section with faces parallel to prismatic planes. This probably results from a predominant role of prismatic glide. The parallel ridges in the fracture surface correspond to the remaining ligaments due to the coalescence of these tubular cavities aligned along a lath boundary. The small dimple zones are associated with the rupture of lath extremities necessary to allow the principle crack to propagate from a colony to the neighboring one; the stress state in these zones is triaxial and activates secondary slip systems resulting in the small rounded dimples.

Cavity nucleation

Cavities can appear either on the prec1p1tates constituting the lath boundaries or inside the lath. Does this mean that two different types of nucleation sites have to be distinguished? Successive polishing of the surface presenting initially a cavity inside a lath, allows one to locate the tubular cavity in the bulk of the sample. It appears that the nucleation had occurred at the lath boundary. Thus, it seems that cavities systematically nucleate on the rows of precipitates constituting the lath boundaries; then the location of a cavity appearing inside a lath in the sample surface is due to the angular difference be tween the tube axis of the cavity, which corresponds to the < c) axis of the lath and the plane of the boundary, which is close to a prismatic plane due to the allotropic transformation [l].

THE HEXAGONAL CAVITY

The main conclusions so far consist of the link between the activation of prismatic slip, principally

(a) (b)
localized in coarse slip bands, and the occurrence of damage. The main damaging effect is due to tubular cavities, which nucleate on precipitates and have an axis parallel to the local < c) axis. The propagation of the cracks is a consequence of the coalescence of these cavities.

In the following, special attention will be paid to the hexagonal cavities (Fig. 5), from the geometrical, mechanical and kinematical point of view.

Experimental characterization

One particular cavity (Fig. 5) and the slip lines surrounding it have been characterized by means of EBSD technique. The determination of the orien- tation of the lath in the sample coordinate system permits one to conclude that the local < c) axis is quasi-perpendicular to the sample surface (87°), which contains the tensile axis and that the faces of the hexagonal cavity are the three prismatic planes. The slip traces, observed around the cavity, are due to the same prismatic planes. It must be noted that the slip traces can be observed because of the imperfect orthogonality of the (c) axis with respect to the sample surface. If the < c) axis was orthogonal to the sample surface, the gliding (a) dislocations should be parallel to the sample surface and consequently should not leave any trace. The slip systems are activated two by two depending on the considered hexagon angle (thus, in a clockwise rotation one can observe the activation of systems 3 and 2, then 3 and I, then I and 2). The Schmid factor of each prismatic slip system has been determined: F1 = 0.49, F2 = 0.16, F3 = 0.33; using a hypothesis of uniform stress. For a distance greater than two or three times the radius of the cavity, but within the same colony, only Systems 1 and 3 are activated (not observable on Fig. 5), the lowest Schmid factor being 0.33 for System 3. The fact that System 2 is solely observed within a distance to the cavity of the order of the radius, although its Schmid factor is weak (F2 = 0.16), suggests that the cavity produces a perturbation of the local stress field.

Moreover a video depicting the nucleation and growth of a cavity was recorded during an in situ tensile test (nominal strain rate Ex of the order 10-4 s-1 ). This video shows that the cavity appears with a rounded shape (0.2 µm) and after few seconds (2 s) takes on an hexagonal shape that remains stable during the deformation with a homothetical growth. The cavity growth rate can be written as:

B = (Ex with ( = 260.

R (I)

Rice and Tracey [5] studied the case of a spherical cavity of radius R growing in material following a Norton-Hoff law (2):

( •)"' a = ao Zo (2)
they obtained:

+(I -m)[l + m(0,4175 + 0,144x(sgn am))] }'' " (3)
with: a m, hydrostatic stress; a,", von Mises equivalent stress; €,", von Mises equivalent strain rate; m, stress sensitivity factor; x = + 1 if the axial stress is greater than the radial stress -1 otherwise.

Also, in the case of uniaxial tension for a Newtonian material (m = 1), this expression gives: R (3)(1Tm).

R. = 4 a €,q.

(4)

McClintock [6] studied the case of a cylindrical cavity of radius R; for a Newtonian (m = 1) material or a perfectly plastic (m = 0) material subjected to a uniaxial tension his model predicts a ( value of order 1 (Fig. 6). Therefore, the experimental cavity growth rate is much faster than predicted by these models.

The experimental observations and contradiction with the prediction pose three questions: Why are the three prismatic slip systems activated two by two around the cavity? Why is the hexagonal shape stable? Why is the cavity growth rate so high?

Stress field analysis

To solve the problem analytically, the calculations performed are for an isotropic elastic solid in plane strain. These geometrical restrictions are consistent with the observation of cavity growth by the sole activation of prismatic slip systems. Consider a circular hole of radius R in an infi nite plate subjected to a remote uniaxial tensile stress S. The stress at any point of the plate in a polar coordinate system (r, B, z) is This stress state is now projected on the three potential prismatic slip systems. If the <c) axis is considered as. the tube axis (in agreement with the obervations) and parallel to the z axis, and if <P is the rotation angle around this axis between the crystal base and the macroscopic base, it follows that: <1 = [�(j3 sin 2</J -cos 2</J )IT12 + Hsin 2</J + J3 cos 2</J )(IT22 -1T11)]; (8) sin

2</J T2 = COS 2</JIT12 + - 2 -(IT 1 1 -IT22); (9)
T3 = [ -Hcos 2</J + J3 sin 2</J )IT 12 with +�(sin 2</J -J3 cos 2</J )(IT22 -1T1 1 )]; (10) IT 11 = ITr cos' (} + ITo sin 2 (} -ITro sin W , (11)

sin W IT 12 = (IT, -ITe)- 2 -+ IT,e COS 2(), ( 1 2)
IT22 = IT, sin 2 (} + ITo cos 2 (} + ITro sin W. ( 13
)
In the test reported here above (Fig. 5) it can be noticed that far from the cavity only Systems 1 and 3 are active; since the lowest Schmid factor of these systems is equal to 0. 33, this value will now be considered as critical for the activation of a system. Thus, the different zones where this criterion is reached can be determined (Fig. 7); Systems I and 3 are activated everywhere except in the pole regions. The perturbation of the applied stress fi eld by the cavity induces areas where System 2 is activated. The different activation zones are in good agreement with the observed slip traces (Fig. 5). The variation of the resolved shear stress of System 2 around the hole (Fig. 7) shows that its sign changes, systematically inducing a centrifugal glide consistent with the growth of the cavity. Another solution of this problem consists of taking a fi nite element approach with an elastic-plastic and kinematical hardening behavior as a constitutive relation. The result of the calculation is presented in Fig. 8 for the same activation criterion as above. The three prismatic slip systems are also activated two by two around the cavity and the stress concentration factors on each of the systems are equal, which means that there is the same quantity of gliding on the three slip systems.

The question about the activation by circular permutation of the three prismatic slip systems fi nds a satisfactory qualitative answer in this simplified approach.

Morphological stability

The stability of the hexagonal morphology can be discussed by analogy between the behavior of each of the six angles of the cavity and the observations made by Neumann [START_REF] Neumann | [END_REF] and Pelloux [START_REF] Pelloux | Proc. Air Force Conj. on Fatigue and Fracture of Aircraft. Structures and Materials[END_REF] of the crack propagation without blunting occurring in a single crystal in double glide under tension. These authors consider that the crack propagates by alternate It ® @ /; activation of the two slip systems concentrated at the crack tip (Fig. 9). This model can be adopted to simulate the growth behavior in each of the six angles by activation of two prismatic slip systems with the same quantity of gliding; this assumption is in agreement with the results of the fi nite element calculation. A simulation is presented in Fig. IO by means of a decomposition in six steps, which correspond to one increment of growth; this kind of representation justifi es the homothetic growth of the cavity. Of course, it must be assumed that a certain amount of hardening on an active slip system induces its disactivation and the activation of another one. That hardening may be due to a dislocation pile-up, induced by the stress gradient (l/r2) around the cavity, which produces a strong decrease in dislocation velocity. If the traces of the slip bands are kept around the cavity and their length is taken equal to the present size of the cavity, the previous simulation conducts to the apparition of triangular patterns that compares favorably to the experimental ones (Fig. 5). Moreover, this kinematical model applied to an initially circular cavity transforms its shape to a hexagon during one increment of growth.

Growth kinetics

In the kinematical model, cavity growth occurs by the creation of steps due to the plastic slip resulting from the nucleation and propagation of dislocations in the vicinity of the hexagon corners. The length (/) of the resulting slip bands can be taken proportion ally to the cavity size (R):

I = aR, with a � I. ( 14
)
The longer the band the larger is the number of dislocations in the corresponding pile-up and the Fig. 9. Model proposed for crack opening under tension by alternate activation of two slip systems [START_REF] Neumann | [END_REF].

larger is the step. If e is the step size, in fi rst approximation: e = /31, (15) with f3 a proportionality factor. For geometrical reasons only, the homothetic growth of the cavity imposes that the shear magnitude in each slip band is y = I near the cavity.

Under the assumptions that the macroscopic Norton-Hoff (2) law applies at the shear band scale: (16) and that the shear stress Yoe far from the cavity is induced by a shear stress -rx, the shear strain at a corner of the cavity under the local shear stress krf{ obeys:

(17)
where k is a stress concentration factor. The time for the formation of one step, i.e. for reaching y = 1, near the cavity is:

y

1 e = y = Yooku m. ( 18
)
One increment of growth for the cavity needs six steps (Fig. 10) and a time !it = 68; during that time the cavity grows by !'!.R = 2e. Then the cavity growth rate R -

3 J3 foo . ( 19
) (20) (21)
The micrograph (Fig. 5) reveals that I :::::: These values inserted in equation (23) give a very high growth rate if the concentration factor k is greater than 1. This case should correspond to the stress state close to the corners of the hexagon. This value is not explicitly determined by an analytical solution, but a fi nite element approach, with a crystalline finite element model, could afford the determination of the concentration factor. However, it seems to be reasonable to take one in the order of 1.2 or 1.3. It follows:

� � 100£00, for k = 1.2 ; �:::::: 3000£00, for k = 1.3.

Then for the cases where the stress sensitivity factor m is weak but nonequal to 0 this kind of model can afford to explain the very fast cavity growth rate (( coefficient) and the stability of the hexagonal shape, observed experimentally.

DISCUSSION

The model presented is based on a description of cavity growth and accounts for crystallographic slip localized in narrow bands, which develop at the corners of the hexagonal cavity. It explicitly accounts for the effect of local stress state near these peculiar points and introduces the material constitutive relation in the growth rate equation. The (coefficient, which characterizes the cavity growth rate (23) is directly proportional to the local stress concentration factor and depends on the stress sensitivity factor m.

In the classical models based on continuum mechanics (e.g. McClintock [6] or Rice and Tracey [5]) the driving force for cavity growth is the global state of stress triaxiality and not the local stress; moreover, in these models there is no real free surface formation since there is not any discontinuity during the cavity growth, two neighboring points remain neighbors. The model presented gives a more physical description of the growth of cavities.

To investigate the difference between these two types of models, two extreme cases are considered: a Newtonian material (m = I) and a perfectly plastic material (m = 0).

A Newtonian material shows a weak stress sensitivity and a growth rate coefficient ( pro portional to the stress concentration factor k, near unity. In that case the crystallographic model gives a growth rate one order of magnitude lower than the continuum mechanics models ( :::::: I as shown in Fig. 6.

A perfectly plastic material shows an infi nite stress sensitivity or in other words no strain hardening. The crystallographic model predicts an infi nite growth rate; this value represents the unstable behavior of a crystal without strain hardening: plastic glide concentrates in a slip line impeding the activation of other systems (except if geometrical hardening is active). Therefore, cavity nucleation is impossible and rupture occurs by concentrated glide. Nevertheless continuum mechanics models predict a ( value still close to unity (( � 1.2).

Thus, the crystallographic approach of cavity growth shows a much stronger sensitivity to the material behavior than the continuum mechanics ones, due to the introduction of the local stress state and of the crystallographic glide. This type of crystallographic approach imposes the knowledge of the constitutive relation of the material and its application to the local scale on each slip system. This is a very important assumption, but for zirconium deformed at room temperature, a "Norton-Hoff" type constitutive relation seems to give a good result. This good result is certainly due to the fact that at room temperature there is only one type of slip system activated, the three prismatic systems.

CONCLUSION

Damage mechanisms in P-treated zirconium are linked to the principal deformation mechanism, prismatic slip. The main damage mechanism is the early nucleation and fast growth of tubular cavities. The slip localization along lath boundaries forces these cavities to form alignments parallel to these boundaries; rupture occurs by crack propagation along the dotted lines formed by the cavities. The axis of the cavities is parallel to the < c > axis of the crystal and, for crystallographic orientations of the lath inducing double glide, these cavities have a hexagonal cross-section. A crystallographic model accounts for the creation of free surface by localization of deformation and explains the hexagonal shape and the very fast cavity growth rate.

Fig. 1 .

 1 Fig. 1. Cavity alignments parallel to the lath boundaries; localization of deformation along the boundary observed thanks to the deformation of fiducial grids.

Fig. 2 .

 2 Fig. 2. Cavity coalescence inside alignments; rupture of the ligament between two neighboring alignments.

Fig. 3 .

 3 Fig. 3. Observation of the rupture surface.

Fig. 4 .

 4 Fig. 4. (a) Hexagonal cross-section of the cavity observed in the sample surface. (b) Tubular dimple due to the cavity observed on Fig. 4(a) after rupture.

Fig. 5 .

 5 Fig. 5. Hexagonal cavity with the three prismatic slip systems (1,2,3) activated two by two (vertical tensile axis).

Fig. 6 .Fig. 7 .

 67 Fig. 6. Comparison between experimental cavity growth rate of zirconium and McClintock prediction for Newtonian (m = I) and perfectly plastic (m = 0) material. O Experimental values.

Fig. 8 .

 8 Fig. 8. Results of a finite element calculation for an elastic-plastic material with kinematical hardening. Left part: representation of the area (shaded) where the criterion is reached on System 3 for a semi-hexagonal cavity. The area of activation of System I is obtained by symmetry with respect to the horizontal axis. Right part: representation of the areas where the criterion is reached on System 2 for a semi-hexagonal cavity.

Fig. 10 .

 10 Fig. 10. Decomposition of a kinematical model based on activation of the three prismatic systems two by two around the cavity with the same amount of gliding.

  and after combination with equations (14) and (15): l!'!.R -! p• k 11 m R!'!.t -3et: Yoo • In the considered geometry: . -�• fx -4 Yx, equation (20) is transformed into !!:_ -4 a {J k lfm .

  5 µm and e :::::: 0.5 µm. So, it is consistent to propose for a = I and f3 = 0.1, and consequently: � = 8 X 10 -2kli mf oc .

  the present model:( = 8 X 10-'kl/ m . (23)Results from the literature [IO] allow one to estimate m to be in the interval: 0.02 ,;:;; m ,;:;; 0.03.

Acknowledgement-The authors wish to thank the CEA/CEREM for supplying both materials and financial support.


João Pedro Ferreira

Job Verdonschot

Ping Wang

Anne Pizard

Timothy Collier

Fozia Z Ahmed

Hans-Peter Brunner-La-Rocca

Andrew L Clark

Franco Cosmi

Joe Cuthbert

Javier Díez

Frank Edelmann

Nicolas Girerd

Arantxa González

Stéphanie Grojean

Mark Hazebroek

Javed Khan

Roberto Latini

Mamas A Mamas

Beatrice Mariottoni

Blerim Mujaj

Pierpaolo Pellicori

Johannes Petutschnigg

; Burkert

Patrick Rossignol

Philippe Rouet

Jan A Staessen

John G F Cleland

Stephane Heymans

& Faiez Zannad

Proteomic and Mechanistic Analysis of Spironolactone in Patients at Risk for HF

Keywords: MPO, myeloperoxidase TFPI, tissue factor pathway inhibitor UPAR, plasminogen activator, urokinase receptor

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Contact to:

Mail: f.zannad@chru-nancy.fr or j.ferreira@chru-nancy.fr

Abstract

Background: Further to their beneficial effects in established heart failure (HF), mineralocorticoid receptor antagonists may act upstream on mechanisms preventing incident HF. In people at risk for developing HF, the "Heart OMics in Aging" (HOMAGE) trial showed that spironolactone treatment could provide antifibrotic and anti-remodelling effects, potentially slowing the progression to HF.

Objectives: To further understand the mechanisms underlying spironolactone effect, we assessed its impact on multiple plasma protein biomarkers and respective underlying biological pathways.

Methods: Baseline, 1-month and 9-months (or last visit), plasma samples of HOMAGE participants were measured for protein biomarkers (n=276) using Olink®Proseek-Multiplex cardiovascular and inflammation panels. Spironolactone effect on biomarkers was assessed by analysis of covariance (ANCOVA) and explored by knowledge-based network analysis.

Results: 527 participants were enrolled, 265 were randomized to spironolactone (25-50 mg/day) and 262 to standard care ("control"). The median (percentile 25-75 ) age was 73 (69-79) years and 26% were female. Spironolactone reduced biomarkers of collagen metabolism (e.g., COL1A1, MMP2), BNP, biomarkers related to metabolic processes (e.g., PAPPA), inflammation and thrombosis (e.g., IL17A, VEGF and urokinase). Spironolactone increased biomarkers that reflect the blockade of the mineralocorticoid receptor (e.g., renin), increased the levels of adipokines involved in anti-inflammatory response (e.g., RARRES2), biomarkers of haemostasis maintenance (e.g., tPA, UPAR), myelosuppressive activity (e.g., CCL16), insulin suppression (e.g., RETN), and inflammatory regulation (e.g., IL12B).

Conclusion:

Proteomic analyses suggest that spironolactone exerts pleiotropic effects including reduction in fibrosis, inflammation, thrombosis, congestion and vascular function improvement, all of which may mediate cardiovascular protective effects potentially slowing progression toward heart failure.

Key-words: spironolactone; heart failure prevention; fibrosis; inflammation; reninangiotensin-aldosterone system

Condensed abstract

We studied the impact of spironolactone on multiple plasma protein biomarkers and respective underlying biological pathways in the HOMAGE trial. Spironolactone reduced biomarkers of collagen metabolism, BNP, biomarkers related to metabolic processes, inflammation and thrombosis. Spironolactone increased biomarkers that reflect the blockade of the mineralocorticoid receptor, increased the levels of adipokines involved in anti-inflammatory response, biomarkers of haemostasis maintenance, myelosuppression, insulin suppression, and inflammatory regulation. Proteomic analyses suggest that spironolactone exerts pleiotropic effects including reduction in fibrosis, inflammation, thrombosis, congestion and vascular function, all of which may mediate cardiovascular protective effects potentially slowing progression toward heart failure. Individuals at risk for developing HF often have higher levels of natriuretic peptides, increased activation of pro-fibrotic, inflammatory and apoptotic pathways, elevation of markers of vascular and endothelial dysfunction, atherosclerosis, and increased activation of the renin-angiotensin-aldosterone system (RAAS) [START_REF] Ferreira | Proteomic Bioprofiles and Mechanistic Pathways of Progression to Heart Failure[END_REF] . Due to their pleiotropic effects beyond sodium and potassium regulation, MRAs (e.g., spironolactone, eplerenone, finerenone) may positively affect these aforementioned mechanisms [START_REF] Pitt | Mineralocorticoid receptor antagonists in patients with heart failure: current experience and future perspectives[END_REF] . In the HOMAGE ("Heart OMics in AGEing") trial, treatment with spironolactone decreased markers of collagen synthesis, decreased the circulating levels of N-terminal pro brain natriuretic peptide (NT-pro BNP), reduced blood pressure and improved cardiac remodelling in asymptomatic people at risk for developing HF (Cleland et al. EHJ 2020 accepted).

Abbreviations list

HF

In this pre-specified secondary analysis of the HOMAGE trial, we aimed to assess the effect of spironolactone on multiple circulating proteomic biomarkers to better characterize the biological pathways that could be affected by spironolactone in individuals at risk of developing HF.

Methods

Trial design and population

The HOMAGE trial was a prospective, randomized, open-label, blinded-endpoint (PROBE), multicentre design, in which people at increased risk of developing HF were randomly assigned to receive either spironolactone or standard of care ("control") -not receiving spironolactone or other MRA (ClinicalTrials.gov Identifier: NCT02556450). The rationale, trial design and main results have been published [START_REF] Pellicori | Effects of spironolactone on serum markers of fibrosis in people at high risk of developing heart failure: rationale, design and baseline characteristics of a proof-of-concept, randomised, precision-medicine, prevention trial. The Heart OMics in AGing (HOMAGE) trial[END_REF] . The study was approved by all relevant ethics committees and regulatory bodies. All participants provided written informed consent prior to study-specific procedures. The main participating criteria included age of 65 or older (amended to 60 years during the course of the trial), cardiovascular risk defined by the presence of coronary artery disease or at least 2 of the following: diabetes, treated hypertension, microalbuminuria, abnormal ECG, and a NT-pro BNP between 125 and 1,000 ng/L or BNP between 35 and 280 ng/L. The main exclusion criteria were glomerular filtration rate (eGFR) <30 mL/minute/1.73m2, serum potassium >5.0 mmol/L, left ventricular ejection fraction <45%, a diagnosis of HF or treatment with loop diuretics and atrial fibrillation/flutter.

HOMAGE trial patients, follow-up and endpoints

A total of 527 patients were randomized (265 to spironolactone and 262 to standard of care). The median (percentile 25-75 ) follow-up time was 8.9 (6.0-9.2) months. The primary endpoint was the interaction between the treatment and the baseline levels of galectin-3 for the change in serum concentrations of PIIINP (procollagen type III N-terminal propeptide) from baseline to the end of follow-up ("9-month visit"). Secondary aims were to

Proteomic biomarkers

Baseline, month 1 and month 9 (or last visit) plasma samples were analysed for 276 protein biomarkers by the TATAA-biocenter using the Olink Proseek® Multiplex cardiovascular (CVD) II, CVD III, and inflammation panels. These panels were selected by the balanced inclusion of proteins with already established associations with cardiovascular disease and HF (e.g., BNP or GDF-15), and many others, more exploratory, with less well-established role (e.g., TRAIL or PAPPA). The proteins were determined using high-throughput Olink Proseek® Multiplex 96x96 kits, which measures 92 manually-selected proteins simultaneously in 1 μl of plasma per kit. Each kit uses a proximity extension assay (PEA) technology with dual-recognition DNA-coupled readout, where 92 oligonucleotide-labeled antibody probe pairs are allowed to bind to their respective target in the sample. The platform provides Log2 normalized protein expression (NPX) values with relative quantification. A detailed description of the Olink® technology is depicted on the website: https://www.olink.com/. The abbreviations, full names and respective Olink® multiplex panels of the studied proteins are described in the Supplemental Table 1. The assays were performed in a blinded fashion to the treatment allocation. The proteomic results were then merged into the database.

Statistical analyses

The primary analysis focused on the changes of the proteins from baseline to month 9 or the final visit (for consistency with the primary report (Cleland et al. EHJ 2020 accepted) using analysis of covariance (ANCOVA) comparing the difference of changes between the control and spironolactone groups in the regression model. A linear regression model was fitted, with the protein change (last visit -baseline) as outcome variable, a binary variable to indicate the treatment group (control/spironolactone), and the baseline protein value (NPX) as covariates. The treatment effect was the coefficient that resulted from the comparison of spironolactone vs. control in the regression model. Residual analysis was used to examine the fit of the model. No data transformation was required to meet the assumptions of linear regression. Similar analyses were performed for the protein change at 1 month. A correction for multiplicity of tests using a false discovery rate q-value <0.05 (FDR5%) as described by Benjamini and Hochberg was applied to the protein change from baseline to the last visit. All the other analyses should be viewed as exploratory, including the interaction term between the treatment and the baseline protein level (below vs. above median) to evaluate if the effect of spironolactone on PIIINP and PICP could have varied by the baseline levels of the studied proteins. Statistical analyses were performed using Stata® (version 16, StataCorp LP).

Bioinformatical and network analyses

The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was used to analyse functional enrichment (GO biological processes, KEGG and Reactome pathways) and connections of proteins that were strongly changed by spironolactone. In addition, we used knowledge-based network analysis with induced network approach by consensuspathDB (CPDB) online server (accessed on 17 February 2020) from Max Planck Institute for Molecular Genetics to identify the links among all significantly changed protein biomarkers, based on known knowledge of interactions (protein interactions, genetic interactions, biochemical interactions, and gene regulatory interactions) [START_REF] Iraqi | Extracellular cardiac matrix biomarkers in patients with acute myocardial infarction complicated by left ventricular dysfunction and heart failure: insights from the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) study[END_REF] . The network analysis also identifies additional proteins limited to the first-degree interactors (intermediate nodes) linking our input proteins (seed nodes), with exclusion of low-confidence interactions and quantified by a z-score ≤20 calculated for each intermediate node. The created network was reorganized in Cytoscape (version 3.7) to cluster proteins based on biological functions and linked this network to spironolactone via mineralocorticoid receptor -aldosterone -angiotensin receptor.

Results

Characteristics of the population

The median (percentile 25-75 ) age was 73 (69-79) years, 26% were female, 71% had coronary artery disease, and 41% diabetes. Supplemental Table 2.

Spironolactone effect on plasma proteins from baseline to the last visit

Compared with the control, 18 proteins decreased with spironolactone treatment at the p<0.05 level, among which 4 proteins strongly decreased at the FDRq<0.05 level. These (FDRq<0.05) were: COL1A1 (collagen type I alpha 1 chain), MMP2 (matrix metalloproteinase 2), BNP (brain natriuretic peptide), and PAPPA (pappalysin 1) (Figure 1 (carboxypeptidase A1), MPO (myeloperoxidase), TFPI (tissue factor pathway inhibitor), UPAR (plasminogen activator, urokinase receptor), TFF3 (trefoil factor 3), CXCL9 (C-X-C motif chemokine ligand 9), ADM (adrenomedullin), KLK6 (kallikrein related peptidase 6), and

PRTN3 (proteinase 3). Table 1 & Figure 2.
The results for all the studied proteins are presented in the supplemental material (Supplemental Tables 3 &4). None of the studied proteins had a strong correlation between each other (Spearman Rho<0.7 for all comparisons; Supplemental Table 5), nor protein-clinical parameter correlation (Spearman Rho<0.5 for all comparisons; Supplemental Table 6). Information regarding network edges, nodes and interactions can be found in Supplemental Table 7.

Proteins that changed both at month 1 and last visit

Compared with control, 19 proteins significantly changed (p<0.05) with spironolactone treatment both at month 1 and month 9. Five proteins decreased: COL1A1, MMP2, BNP, VEGFD, and NOTCH3; and 14 proteins increased: REN, IL12B, AMBP, CCL19, CCL25, TRAIL, CSTB, FABP4, TNFRSF9, CST5, CCL3, CPA1, TFF3, CXCL9. Table 2.

Network analysis

The 10 proteins which strongly changed after spironolactone treatment (FDRq<0.05) were used as input to create a focused network. Overall, three clusters of proteins can be identified, collectively fitting six biological functions (Figure 1). The pathways with the strongest associations with the 10 proteins are the RAAS-pathway, extracellular matrix (ECM) metabolism, insulin growth factor (IGF) signalling, haemostasis and adipocytokine signalling.

To get a broader overall picture of the biological network, we used all the 51 proteins that significantly changed with spironolactone treatment (p<0.05) to generate

Proteins that could influence the effect of spironolactone on collagen

We tested the interaction between all the baseline proteins (below vs. above the median) and the effect of spironolactone on PICP and PIIINP. For PICP the strongest interaction was with AXL (AXL receptor tyrosine kinase), whereby AXL levels above the median could predict a major PICP reduction by spironolactone, with no spironolactone effect with AXL levels below the median (p for interaction =0.005). For PIIINP the strongest interaction was with CCL28 (C-C motif chemokine ligand 28), whereby CCL28 levels below the median could predict a major PIIINP reduction by spironolactone, with no spironolactone effect with CCL28 levels above median (p for interaction =0.003). Supplemental Table 8. However, all the studied interactions were statistically non-significant when corrected for multiple testing at a FDRq level <0.05.

Discussion

To the best of our knowledge this is the first report showing a MRA effect on a large panel of circulating proteins. This study confirms that spironolactone has a major effect in reducing markers of collagen metabolism (e.g., COL1A1, MMP2), likely reflecting its anti-fibrotic effects, and reduces BNP the single most important prognostic marker of adverse cardiovascular events. Moreover, spironolactone reduces markers related to metabolic processes (e.g., PAPPA), inflammation and thrombosis (e.g., IL17A, VEGF and urokinase), and increases levels of adipokines involved in white adipose tissue formation and antiinflammatory response (e.g., RARRES2), and markers of haemostasis maintenance (e.g., VWF), myelosuppressive activity (e.g., CCL16), insulin suppression (e.g., RETN), and inflammatory regulation (e.g., IL12B). On the other hand, expectedly, spironolactone increases markers that reflect the blockade of the mineralocorticoid receptor (e.g., renin) Spironolactone has been used for over 50 years. However, until around 20 years ago, spironolactone was thought to be mainly a potassium-sparing diuretic. Increasing evidence shows that the mineralocorticoid receptor (MR) is expressed in the vascular smooth muscle, endothelial cells, and macrophages, myocardium, kidney, brain, bone and several other tissues (e.g., intestines and eyes), has led to an intense investigation on the role of the MR and its blockade, especially after the publication of RALES (the effect of spironolactone on morbidity and mortality in patients with severe heart failure) [START_REF] Jaisser | Emerging Roles of the Mineralocorticoid Receptor in Pathology: Toward New Paradigms in Clinical Pharmacology[END_REF][START_REF] Pitt | The effect of spironolactone on morbidity and mortality in patients with severe heart failure. Randomized Aldactone Evaluation Study Investigators[END_REF] . Particularly interesting was the observation that spironolactone could decrease markers of collagen synthesis, that were associated with adverse cardiac remodelling and poor prognosis in severe HF [START_REF] Zannad | Limitation of excessive extracellular matrix turnover may contribute to survival benefit of spironolactone therapy in patients with congestive heart failure: insights from the randomized aldactone evaluation study (RALES). Rales Investigators[END_REF] . Later these findings were also replicated with eplerenone in other patient-populations [START_REF] Iraqi | Extracellular cardiac matrix biomarkers in patients with acute myocardial infarction complicated by left ventricular dysfunction and heart failure: insights from the Eplerenone Post-Acute Myocardial Infarction Heart Failure Efficacy and Survival Study (EPHESUS) study[END_REF][START_REF] Ferreira | Effect of eplerenone on extracellular cardiac matrix biomarkers in patients with acute ST-elevation myocardial infarction without heart failure: insights from the randomized double-blind REMINDER Study[END_REF][START_REF] Stienen | Eplerenone prevents an increase in serum carboxyterminal propeptide of procollagen type I after myocardial infarction complicated by left ventricular dysfunction and/or heart failure[END_REF] . The MR can be activated by aldosterone and cortisol in the setting of "neurohormonal activation". Once activated, the MRs are associated with a number of cascade effects including the increase in reactive oxygen species, decrease in nitric oxide availability, increase in inflammatory cytokines, insulin resistance, activation and infiltration of macrophages, sodium retention and potassium loss, as well as an increase in systemic fibrosis leading to organ dysfunction and death [START_REF] Vecchiola | Aldosterone Production and Signaling Dysregulation in Obesity[END_REF][START_REF] Demarco | Low-Dose Mineralocorticoid Receptor Blockade Prevents Western Diet-Induced Arterial Stiffening in Female Mice[END_REF] .

In the HOMAGE trial, treatment with spironolactone did not reduce the circulating levels of PIIINP, but reduced PICP and NT-pro BNP while also increasing CITP, suggesting a favourable effect in collagen turnover (with decreased synthesis and increased degradation) and improvement in cardiac remodelling (also supported by the improvement of several echocardiographic parameters (Cleland et al. EHJ 2020 accepted). Spironolactone did increase the levels of galectin-3 (a finding also reported in the Aldo-DHF trial) [START_REF] Edelmann | Galectin-3 in patients with heart failure with preserved ejection fraction: results from the Aldo-DHF trial[END_REF] . Galectin-3 is one mediator of the fibrotic effect of aldosterone and an increase in galectin-3 production, might have been mediated by a rise in aldosterone in consequence of the MR blockade (i.e., a feedback mechanism as discussed above) [START_REF] Lin | Aldosterone induced galectin-3 secretion in vitro and in vivo: from cells to humans[END_REF] . As the MR is present in many tissues it is likely that its blockade influences multiple biological pathways and pathophysiological mechanisms. In this regard, the present study provides important mechanistic insights.

Collagen type I alpha 1 chain (COL1A1), MMP2, BNP, and PAPPA were decreased by spironolactone. Collagen type I and BNP reflect the reduction in collagen synthesis and reverse cardiac remodelling, respectively; and their reduction is consistent with previously published findings using other procedures and biomarkers that reflect the same pathways (Cleland et al. EHJ 2020 accepted), thus reinforcing the robustness of our results. Both COL1A1 and BNP, as well as MMP2, were reduced after 1 month of treatment, suggesting that the effect of spironolactone on the ECM/collagen formation and cardiac remodelling occurs early after its administration and is sustained over time. By modulating collagen, MMP2 regulates the ECM, bone formation, tissue repair, angiogenesis and tumor invasion [START_REF] Martignetti | Mutation of the matrix metalloproteinase 2 gene (MMP2) causes a multicentric osteolysis and arthritis syndrome[END_REF] .

MMP-2 may contribute to perpetuating the pro-fibrotic response by generating matrikines in the process of collagen degradation, as wells as by activating growth factors (e.g., transforming growth factor-β), which in turn induce collagen synthesis. Indeed, targeted deletion of MMP-2 reduced the development of myocardial fibrosis and improve hypertension-induced cardiac hypertrophy in mice with chronic pressure overload [START_REF] Mendes | Verapamil decreases calpain-1 and matrix metalloproteinase-2 activities and improves hypertension-induced hypertrophic cardiac remodeling in rats[END_REF] . MMP2 also acts on several non-matrix proteins such as big endothelin-1 and beta-type calcitoningene related peptide, which promotes vasoconstriction [START_REF] Abdalvand | Matrix metalloproteinase enhances big-endothelin-1 constriction in mesenteric vessels of pregnant rats with reduced uterine blood flow[END_REF] . The metalloproteinase further increases myocardial oxidative stress and regulate myocardial cell death pathways. The reduction of MMP2 by spironolactone supports the potential beneficial effects of the drug in reducing vasoconstriction, oxidative stress and cardiac cell death [START_REF] Yarbrough | Caspase inhibition modulates left ventricular remodeling following myocardial infarction through cellular and extracellular mechanisms[END_REF] . Furthermore, MMP2 plays a role in the IGF pathway by cleaving IGF binding proteins (IGFBP). Pappalysin-A or pregnancy-associated plasma protein-A (PAPPA), is a metalloproteinase that also cleaves IGFBP. In animal models, the deletion of PAPPA increased circulating IGFBP-5 levels and was associated with a reduction in the collagen markers of bone turnover [START_REF] Christians | Pappa2 deletion has sex-and age-specific effects on bone in mice[END_REF] ; it is thus possible that the reduction of PAPPA is related to the reduction of collagen markers.

Vascular endothelial growth factor D (VEGFD) and NOTCH3 were also reduced after 1-month spironolactone treatment and remained reduced until the end of the study (despite not passing the multiple test correction threshold). VEGFD plays an active role in angiogenesis, lymphatic angiogenesis, and endothelial cell growth [START_REF] Leppanen | Structural determinants of vascular endothelial growth factor-D receptor binding and specificity[END_REF] . In patients with agerelated macular degeneration refractory to anti-VEGF treatment, spironolactone could reduce neovascularization, suggesting that spironolactone might be able to modulate angiogenesis [START_REF] Zhao | Mineralocorticoid receptor antagonism limits experimental choroidal neovascularization and structural changes associated with neovascular age-related macular degeneration[END_REF] . NOTCH3 interferes with cell proliferation and apoptotic programs. NOTCH3 has been found to be upregulated in some tumor tissues and may contribute to premature biological ageing [START_REF] Peters | CADASIL-associated Notch3 mutations have differential effects both on ligand binding and ligand-induced Notch3 receptor signaling through RBP-Jk[END_REF] . Whether spironolactone can delay these processes is worth investigating.

MRAs increase renin and aldosterone by a feedback mechanism after blockade of the MR. Thus, in the setting of MRA use, the measurements of renin and aldosterone have limited clinical utility [START_REF] Kobayashi | Clinical determinants and prognostic implications of renin and aldosterone in patients with symptomatic heart failure[END_REF] . In our study, and as part of normal physiological feedback mechanisms, renin was increased by spironolactone, which shows drug compliance.

Retinoic acid receptor responder 2 (RARRES2) or chemerin is an adipokine that regulates adipogenesis in a process associated with the expansion of white adipose tissue, and may have anti-inflammatory properties via increased production of nitric oxide [START_REF] Yamawaki | A novel adipocytokine, chemerin exerts anti-inflammatory roles in human vascular endothelial cells[END_REF] .

Furthermore, RARRES2 results from the cleavage of pre-chemerin by proteases involved in fibrinolysis, and fibrinolysis is a major pathway in our extended network. Von Willebrand factor (VWF) is important for the maintenance of haemostasis as it promotes the adhesion of platelets to the sites of vascular injury by forming a molecular bridge between subendothelial collagen matrix and platelets. Spironolactone enhances collagen degradation, which may contribute to increased plasma free form of VWF. In addition, several proteins involved in fibrinolysis where elevated after 9 months of spironolactone treatment: tissue plasminogen activator (PLAT), plasminogen activator (PLAUR) and tissue factor pathway inhibitor (TFPI). As aldosterone promotes haemostasis by enhancing angiotensin II-induced plasminogen activator inhibitor (PAI-1), spironolactone may inhibit haemostasis and promote clot dissolution. The proteomic profile shows that spironolactone has a strong mediating role in pro-vasodilation, inhibiting platelet adhesion, suppression of coagulation and fibrinolysis; implying an anti-thrombotic potential. Overexpression of CCL16 reduced the collagen content in hepatic fibrotic cell lines. CCL16 also has potent myelosuppressive activity, and suppresses the proliferation of myeloid progenitor cells [START_REF] Youn | Isolation and characterization of LMC, a novel lymphocyte and monocyte chemoattractant human CC chemokine, with myelosuppressive activity[END_REF] . Thus, the increase in the circulating levels of CCL16 may be one of the mechanisms by which spironolactone both reduces collagen content and exerts a mild myelosuppressive activity. Patients taking spironolactone tended to have a lower haemoglobin concentration, while also having lower weight (Cleland et al. EHJ 2020 accepted). Resistin (RETN) has been found to be increased in the context of a low sodium diet where RAAS activity is physiologically high [START_REF] Vaidya | The association of plasma resistin with dietary sodium manipulation, the renin-angiotensin-aldosterone system, and 25-hydroxyvitamin D3 in human hypertension[END_REF] ; a condition that is similar to that induced by MRAs. Interleukin-12 beta subunit (IL12B) is an inflammatory cytokine that has been associated with chronic kidney disease and renal function [START_REF] Fan | Up-regulation of tubular epithelial interleukin-12 in autoimmune MRL-Fas(lpr) mice with renal injury[END_REF] ; it is possible that the rise in this cytokine may be associated with the slight rises in creatinine and galectin-3 with spironolactone treatment (Cleland et al. EHJ 2020 accepted). This hypothesis is supported by the positive, albeit weak, correlation between IL12B and creatinine.

The central "hub" of our extended network was the angiotensin receptor nonspecific effector SRC, which is a tyrosine kinase, involved in several biological process, including immune response, cell adhesion, cell cycle progression, apoptosis, migration, and transformation. The SCR pathway is an important effector of angiotensin receptor which controls aldosterone formation [START_REF] Mehta | Angiotensin II cell signaling: physiological and pathological effects in the cardiovascular system[END_REF] . Our extended network shows a central role for SRC in mediating all effects of spironolactone on the proteomic profile: the immune response, cell adhesion, haemostasis and fibrinolysis, and ECM metabolism. Thus, inhibiting the effects of aldosterone may initiate a cascade of downstream pathway mediated by SRC, which helps explaining the pleiotropic effects of spironolactone.

Limitations

We tested the effect of spironolactone on multiple proteins applying a correction for test multiplicity to limit the occurrence of false positive findings; however, as HOMAGE was a randomized controlled trial, other proteins, which levels were also significantly changed with spironolactone, might also be implicated in relevant pathways and biological processes and could be worth exploring in further studies. Additionally, many of the highlighted mechanisms should be furtherly replicated and confirmed at a cellular level. We measured three Olink® panels (CVDII, CVDIII, and inflammation), and measuring more proteins could have provided further insight on the mechanisms of action and effects of spironolactone.

However, the hypothesis is that spironolactone would mainly influence processes associated to cardiovascular and inflammatory processes.

Conclusion

Proteomic analyses suggest that spironolactone may have a pleiotropic mode of action that includes the reduction of biomarkers associated with fibrosis, congestion, inflammation, and vascular function. Additional metabolic and potentially anti-apoptotic effects could also be found. Our proteomic approach for examining the underlying mechanisms of MRA action confirms, for the first time in a clinical setting, many results that had been only described in experimental models. This highlights the usefulness of proteomics for pathophysiology and pharmacology clinical investigations.

Perspectives

Core Clinical Competencies

Spironolactone may delay the heart failure onset in people with high cardiovascular risk. The effects of spironolactone are mediated by a reduction in pro-fibrotic factors, but also by reducing inflammation, improving angiogenesis and metabolism.

Translational Outlook

Spironolactone reduced biomarkers of collagen metabolism (e.g., COL1A1, MMP2), BNP, biomarkers related to metabolic processes (e.g., PAPPA), inflammation and thrombosis (e.g., IL17A, VEGF and urokinase). Spironolactone increased biomarkers that reflect the blockade of the mineralocorticoid receptor (e.g., renin), increased the levels of adipokines involved in anti-inflammatory response (e.g., RARRES2), biomarkers of haemostasis maintenance (e.g., tPA, UPAR), myelosuppressive activity (e.g., CCL16), insulin suppression (e.g., RETN), and inflammatory regulation (e.g., IL12B). These biomarkers and pathways could be further tested as potential therapeutic targets.

  Faiez Zannad or Dr. João Pedro Ferreira Centre d'Investigation Clinique 1433 module Plurithématique, CHRU Nancy -Hopitaux de Brabois Institut Lorrain du Coeur et des Vaisseaux Louis Mathieu 4 rue du Morvan, 54500 Vandoeuvre les Nancy Tel : +33 (0) 3 83 15 73 15 Fax : +33 (0) 3 83 15 73 24

  , heart failure MRA, mineralocorticoid receptor antagonist RAAS, renin-angiotensin-aldosterone system COL1A1, collagen type I alpha 1 chain MMP2, matrix metalloproteinase 2 BNP, brain natriuretic peptide PAPPA, pappalysin 1 VEGFD, vascular endothelial growth factor D NOTCH3, notch receptor 3 EPCAM, epithelial cell adhesion molecule BOC, BOC cell adhesion associated oncogene regulated IL4RA, interleukin 4 receptor subunit alpha IL17A-1-microglobulin/bikunin precursor CCL19, C-C motif chemokine ligand 19 MMP7, matrix metalloproteinase 7 PLC, phospholipase C gamma 1 CCL25, C-C motif chemokine ligand 25 TRAIL, TRAIL/TNF superfamily member 10 TPA, tissue-type plasminogen activator GAL9, galectin-9 NT3, neurotrophin 3 SRC, SRC proto-oncogene, non-receptor tyrosine kinase CSTB, cystatin B FABP4, fatty acid binding protein 4 GDF15, growth differentiation factor 15 TNFRSF9, TNF receptor superfamily member 9 CST5, cystatin D CCL3, C-C motif chemokine ligand 3 CPA1, carboxypeptidase A1 Introduction Preventing, rather than treating HF, might be a more effective way of increasing long-term survival and delay health-related quality of life impairment. Further to their beneficial effects in established HF, mineralocorticoid receptor antagonists (MRAs) might be useful for preventing incident HF 1 .

  investigate the effects of spironolactone on the change (from baseline to the end of followup) of other markers of collagen metabolism (procollagen type I C-terminal propeptide [PICP] and collagen type I-C terminal telopeptide [CITP]), NT-pro BNP, echocardiographic measures of cardiac structure and function, and signs/symptoms. One-month changes were also assessed in exploratory analyses. At baseline, one month and at the end of the study, participants performed clinical, biomarker and echocardiographic measurements (Cleland et al. EHJ 2020 accepted).

2 .

 2 ); plus (p<0.05) VEGFD (vascular endothelial growth factor D), NOTCH3 (notch receptor 3), EPCAM (epithelial cell adhesion molecule), BOC (BOC cell adhesion associated, oncogene regulated), IL4RA (interleukin 4 receptor subunit alpha), IL17A (interleukin 17A), SELE (selectin E), APN (aminopeptidase N), THBS2 (thrombospondin 2), AXL (AXL receptor tyrosine kinase), TIE2 (angiopoietin-1 receptor), ALCAM (activated leukocyte cell adhesion molecule), CNTN1 (contactin 1), and IL17D (interleukin17D). Table 1 & Figure Compared with control, 33 proteins increased with spironolactone treatment at the p<0.05 level, among which 6 proteins strongly increased at the FDRq<0.05 level. These (FDRq<0.05) were: REN (renin), RARRES2 (retinoic acid receptor responder 2), VWF (Von Willebrand factor), CCL16 (C-C motif chemokine ligand 16), RETN (resistin), and IL12B (interleukin 12B) (Figure 1); plus (p<0.05) PGLYRP1 (peptidoglycan recognition protein 1), IL6RA (interleukin 6 receptor A), AMBP (alpha-1-microglobulin/bikunin precursor), CCL19 (C-C motif chemokine ligand 19), MMP7 (matrix metallopeptidase 7), PLC (phospholipase C gamma 1), CCL25 (C-C motif chemokine ligand 25), TRAIL (TRAIL/TNF superfamily member 10), TPA (tissue-type plasminogen activator), GAL9 (galectin-9), NT3 (neurotrophin 3), SRC (SRC proto-oncogene, non-receptor tyrosine kinase), CSTB (cystatin B), FABP4 (fatty acid binding protein 4), GDF15 (growth differentiation factor 15), TNFRSF9 (TNF receptor superfamily member 9), CST5 (cystatin D), CCL3 (C-C motif chemokine ligand 3), CPA1

Figure 2 .

 2 Figure 2. Spironolactone directly affected the RAAS-pathway and then activate downstream

(

  brain natriuretic peptide), PAPPA (pappalysin 1), VEGFD (vascular endothelial growth factor D), NOTCH3 (notch receptor 3), EPCAM (epithelial cell adhesion molecule), BOC (BOC cell adhesion associated, oncogene regulated), IL4RA (interleukin 4 receptor subunit alpha), IL17A (interleukin 17A), SELE (selectin E), APN (aminopeptidase N), THBS2 (thrombospondin 2), AXL (AXL receptor tyrosine kinase), TIE2 (angiopoietin-1 receptor), ALCAM (activated leukocyte cell adhesion molecule), CNTN1 (contactin 1), IL17D (interleukin17D), REN (renin), RARRES2 (retinoic acid receptor responder 2), VWF (Von Willebrand factor), CCL16 (C-C motif chemokine ligand 16), RETN (resistin), IL12B (interleukin 12B), PGLYRP1 (peptidoglycan recognition protein 1), IL6RA (interleukin 6 receptor A), AMBP (alpha-1microglobulin/bikunin precursor), CCL19 (C-C motif chemokine ligand 19), MMP7 (matrix metalloproteinase 7), PLC (phospholipase C gamma 1), CCL25 (C-C motif chemokine ligand 25), TRAIL (TRAIL/TNF superfamily member 10), TPA (tissue-type plasminogen activator), GAL9 (galectin-9), NT3 (neurotrophin 3), SRC (SRC proto-oncogene, non-receptor tyrosine kinase), CSTB (cystatin B), FABP4 (fatty acid binding protein 4), GDF15 (growth differentiation factor 15), TNFRSF9 (TNF receptor superfamily member 9), CST5 (cystatin D), CCL3 (C-C motif chemokine ligand 3), CPA1 (carboxypeptidase A1), MPO (myeloperoxidase), TFPI (tissue factor pathway inhibitor), UPAR (plasminogen activator, urokinase receptor), TFF3 (trefoil factor 3), CXCL9 (C-X-C motif chemokine ligand 9), ADM (adrenomedullin), KLK6 (kallikrein related peptidase 6), PRTN3 (proteinase 3).

  (collagen type I alpha 1 chain), MMP2 (matrix metalloproteinase 2), BNP (brain natriuretic peptide), VEGFD (vascular endothelial growth factor D), NOTCH3 (notch receptor 3), REN (renin), IL12B (interleukin 12B), AMBP (alpha-1-microglobulin/bikunin precursor), CCL19 (C-C motif chemokine ligand 19), CCL25 (C-C motif chemokine ligand 25), TRAIL (TRAIL/TNF superfamily member 10), CSTB (cystatin B), FABP4 (fatty acid binding protein 4), TNFRSF9 (TNF receptor superfamily member 9), CST5 (cystatin D), CCL3 (C-C motif chemokine ligand 3), CPA1 (carboxypeptidase A1), TFF3 (trefoil factor 3), CXCL9 (C-X-C motif chemokine ligand 9).

Figure 1 .

 1 Figure 1. Focused network of biomarkers influenced by spironolactone treatment from

Figure 2 .

 2 Figure 2. Extended network for the biomarkers that changed with spironolactone treatment

Table 1 .

 1 Changes in biomarkers with spironolactone treatment (from baseline to the last

	visit)
	Biomarker	β-coef. (95%CI)	P-value	FDRq
	Decreased with spironolactone
	COL1A1	-0.14 (-0.20 to -0.08)	0.00001	0.0014
	MMP2	-0.10 (-0.16 to -0.04)	0.0008	0.032
	BNP	-0.28 (-0.44 to -0.11)	0.001	0.041
	PAPPA	-0.13 (-0.20 to -0.05)	0.001	0.044
	VEGFD	-0.08 (-0.13 to -0.03)	0.003	0.064
	NOTCH3	-0.09 (-0.15 to -0.03)	0.003	0.068
	EPCAM	-0.16 (-0.27 to -0.05)	0.005	0.090
	BOC	-0.06 (-0.11 to -0.02)	0.007	0.10
	IL4RA	-0.09 (-0.15 to -0.02)	0.007	0.10
	IL17A	-0.15 (-0.27 to -0.04)	0.009	0.12
	SELE	-0.09 (-0.16 to -0.02)	0.011	0.12
	APN	-0.07 (-0.12 to -0.01)	0.013	0.13
	THBS2	-0.04 (-0.08 to -0.01)	0.019	0.17
	AXL	-0.07 (-0.13 to -0.01)	0.027	0.20
	TIE2	-0.05 (-0.09 to -0.01)	0.027	0.20
	ALCAM	-0.06 (-0.11 to -0.01)	0.027	0.20
	CNTN1	-0.06 (-0.12 to -0.01)	0.033	0.23
	IL17D	-0.06 (-0.11 to -0.01)	0.037	0.24
	Increased with spironolactone

Table 2 .

 2 Biomarkers that changed both at month 1 and last visit

		Month 1		Last visit
	Biomarker
		β-coef. (95%CI)	P-value	β-coef. (95%CI)	P-value
	Decreased with spironolactone
	COL1A1	-0.08 (-0.13 to -0.02)	0.0043	-0.14 (-0.2--0.08)	0.00001
	MMP2	-0.10 (-0.16 to -0.04)	0.0008	-0.1 (-0.16--0.04)	0.0008
	BNP	-0.46 (-0.61 to -0.32)	0.00001	-0.28 (-0.44--0.11)	0.0012
	VEGFD	-0.09 (-0.14 to -0.04)	0.0003	-0.08 (-0.13--0.03)	0.0028
	NOTCH3	-0.08 (-0.13 to -0.02)	0.0094	-0.09 (-0.15--0.03)	0.0032
	Increased with spironolactone
	REN	0.48 (0.38-0.58)	0.00001	0.53 (0.42-0.63)	0.00001
	IL12B	0.10 (0.03-0.17)	0.0058	0.13 (0.05-0.22)	0.0016
	AMBP	0.05 (0.02-0.08)	0.0029	0.05 (0.01-0.08)	0.0058
	CCL19	0.12 (0.03-0.21)	0.0063	0.15 (0.04-0.26)	0.0071
	CCL25	0.10 (0.04-0.15)	0.0007	0.07 (0.02-0.13)	0.010
	TRAIL	0.05 (0.01-0.10)	0.042	0.06 (0.01-0.11)	0.011
	CSTB	0.13 (0.04-0.21)	0.0033	0.1 (0.02-0.19)	0.021
	FABP4	0.12 (0.03-0.22)	0.010	0.12 (0.02-0.23)	0.024
	TNFRSF9	0.11 (0.05-0.17)	0.0007	0.08 (0.01-0.15)	0.027
	CST5	0.09 (0.03-0.15)	0.0019	0.07 (0.01-0.13)	0.033
	CCL3	0.09 (0.01-0.17)	0.027	0.09 (0.01-0.18)	0.034
	CPA1	0.13 (0.02-0.23)	0.016	0.12 (0.01-0.22)	0.035
	TFF3	0.07 (0.01-0.13)	0.022	0.07 (0-0.13)	0.042

Table 1 .

 1 The full names of intermediates are depicted in Supplemental Table8. Additionally, 3 collagen metabolism plasma biomarkers (PICP, CITP and PIIINP) are also included in this network.

The authors have no relevant conflicts of interest to disclose with regards to the content of this manuscript.

Bibliography

Funding HOMAGE was funded by a grant from the European Union 7th Framework Programme for Research and Technological Development (HEALTH-F7-305507 HOMAGE (EU FP7 305507 http://www.homage-hf.eu). JPF, PR, NG, FZ are supported by the RHU Fight-HF, a public grant overseen by the French National Research Agency (ANR) as part of the second "Investissements d'Avenir" program (reference: ANR-15-RHUS-0004), by the French PIA project "Lorraine Université d'Excellence" (reference: ANR-15-IDEX-04-LUE), Contrat de Plan Etat Lorraine IT2MP and FEDER Lorraine.

Disclosures

Central Illustration. Overall integration of the spironolactone effects on the proteomic profile of people at risk for developing heart failure.

Caption: This figure summarizes the main effects of spironolactone in the circulating proteins measured the HOMAGE trial.


Nicolas Probst

Dr Rémy Lartia

Prof Océane E Théry

Mouâd Alami

Eric Defrancq
email: eric.defrancq@univ-grenoble-alpes.fr

Samir Messaoudi
email: samir.messaoudi@u-psud.fr

Drs N Probst

Defrancq

Keywords: Oligonucleotides, thioglycoconjugation, Pd-catalysis, thiosaccharide, DNA-glycoconjugates

An efficient method for the thioglycoconjugation of iodinated oligonucleotides by Buchwald-Hartwig-Migita crosscoupling under mild conditions is reported. The method enables divergent synthesis of many different functionalized thioglycosylated ODNs in good yields, without affecting the integrity of the other A, C and G nucleobases.

Synthetic oligonucleotides are currently investigated for various applications in medical research (e.g. control of gene expression), [1] molecular diagnostic (e.g. DNA-based biosensors) [2] as well as in biotechnology (e.g. catalysts) [3] and nanotechnology (e.g. origami DNA for nanomaterials) [4] . Tremendous efforts have been made to improve the intrinsic properties of oligonucleotides by either incorporating chemical modifications in their structure (e.g phosphorothioate, 2'methoxyethoxy, locked nucleic acids…) or by covalently anchoring reporter groups with relevant properties to them. The conjugation with a reporter group is of great interest because it can be used not only to improve the existing oligonucleotide properties but also to impart it entirely new properties. [5] In this context several approaches including the incorporation of modified phosphoramidites, step-wise (in-line) solid-phase synthesis, on-support fragment coupling, and solution-phase coupling, have been developed so far. [6] Whereas on-support conjugation is convenient for the incorporation of a variety of reporters due to commercially available building blocks, it shows some limitations including the need for the synthesis of appropriate phosphoramidite building blocks for the introduction of non-commercially available modifications and the stability of the desired reporter during automated DNA synthesis and subsequent deprotection, the latter being carried out in drastic basic conditions. Furthermore the incorporation of bulky building block could hamper the efficiency of the phosphoramidite coupling reaction. Enzymatic incorporation of modified bases can circumvent those stability issues but polymerase-catalyzed synthesis of modified DNA could be sensitive to the incorporated reporter groups and site-selective incorporation of a single modified base is difficult to achieve. [START_REF]Polymerase Synthesis of Base-Modified DNA[END_REF] Post-synthetic approaches can overcome these limitations and are the most extensively used for oligonucleotide conjugation. For this purpose, a number of chemical linkages have been developed by introducing a reactive moiety onto nucleic acid for subsequent coupling reaction with reporter bearing complementary reactive group. In this context, efficient strategies involving oxime [8] and thiol-maleimide [9] conjugations, Cu-catalyzed [10] (or Cu-free) [11] azide-alkyne cycloaddition, Staudinger ligation [START_REF] Arthur | Etheve-Quelquejeu in Chemistry of Organo-Hybrids: Synthesis and Characterization of Functional Nano-Objects[END_REF] as well as Diels-Alder reactions [13] have been reported. Another elegant method to conjugate oligonucleotides is the late stage diversification from a common halogenated oligonucleotide precursor under palladium catalysis. [14] The first post-functionalization has been reported by Manderville who described Suzuki-Miyaura cross-coupling reaction at C-8 position of a guanine nucleobase from an oligonucleotide containing a single 8-bromo-2'-deoxyguanosine residue (Scheme 1). [15] Extend to Stille coupling reaction has been next achieved by Jäschke who reported the efficient reaction of triorganotin compounds with iodine functionalized oligonucleotides and nucleotide triphosphates. [16] Challenging substrates, like hindered boronic acids, have been incorporated by Suzuki-Miyaura cross-coupling reaction for designing photoswitchable nucleotides inside a duplex DNA. [17] Interestingly, sensitive and useful functional groups were successfully incorporated by Davis using Suzuki-Miyaura cross-coupling reaction to 5-iodouridine under mild conditions. [18] The highvalue of Pd-catalyzed bond formation is just beginning to reveal useful applications in biological systems as emphasized by recent results. [19] However, to date, only C-C cross-coupling reactions (Suzuki, Stille and Sonogashira) of halogenated oligonucleotides have been reported and further development of these methodologies for the introduction of other useful groups such as sugars is of great interest. Carbohydrates are indeed involved in many biological events and play crucial roles in various cellular recognition processes. [20] A better understanding of their interactions with proteins is a major issue for the development of new therapies. Recently, Winssinger [21] reported a dynamic cooperative glycan assembly of PNA-fucose conjugates and the biomolecular interaction between glucose and nucleobase has been demonstrated by Morales [22] showing Efficient Buchwald-Hartwig-Migita cross-coupling for DNA thioglycoconjugation the benefits of carbohydrate incorporation on oligomeric structures. Several strategies for coupling carbohydrates to oligonucleotides are reported in the literature, [23] and in all cases the sugar moiety is attached to the oligonucleotide through an adapted linker (platform). To the best of our knowledge there is no report concerning the direct coupling of carbohydrates on the nucleobase, that has motivated our study for the postfunctionalization of halogenated oligonucleotides with thiosugars under Pd-catalysis. In our efforts to functionalize sugars under transition-metal catalysis, [24] our group recently reported an efficient protocol for the palladium-catalyzed coupling of aryl and alkenyl halides with various α-and β-glycosyl thiols. [25] The C-S bond-forming reaction was rapidly achieved (5 min) at room temperature by using G3-XantPhos palladacycle pre-catalyst [26] (1 mol%), in the presence of Et3N (1.0 equiv) in THF. We envisioned in the present study whether 5-iodo 2'-deoxyuridine (5-IdU) moiety could be utilized as building blocks for the synthesis of a range of thioglyco-oligonucleotides (Scheme 1). We demonstrate herein that the palladium-catalyzed thioglyconjugation is compatible with DNA and is applicable to a wide variety of oligonucleotides (i.e. with different lengths and containing purines and pyrimidines nucleobases) and carbohydrates derivatives.

To establish the appropriate conditions for the coupling reaction, tetra-O-acetylated 1-thio-β-D-glucopyranose 1a and unprotected 5-iodouridine 2 were initially selected as coupling model substrates (for the optimization of the reaction conditions see SI). A full conversion and 95% yield of isolated product 3a was obtained using 0.1 equiv of G3-XantPhos catalyst (Scheme 2). Importantly, the slow addition of thiosugar (for ~ 1 h) proved efficient and limited the formation of thiosugar dimer byproduct. [START_REF]Copper-mediated arylsulfanylations of pyrimidine nucleosides and nucleotides have been recently reported[END_REF] Motivated by this result, we next explored the scope of the Scheme 2. Palladium-catalyzed thioglycoconjugation of 5-iodouridine 2. Reaction conditions: thiosaccharide (1.2 equiv) in THF (0.7 mL) added over 1 hour via a syringe pump to a solution of 5-iodouridine (0.07 mmol, 1 equiv), G3-XantPhos (10 mol-%), TEA (1.3 equiv) in H2O/THF (0.7 mL/0.1 mL) at 60 °C. a yield of isolated product. b direct addition of the thiosugar.

reaction by coupling on nucleoside 2, structurally diverse βmono-, di-and poly-thiosugar derivatives 1a-g (Scheme 2). Gratifyingly, all the coupling reactions proceeded in good yields and without epimerisation at anomeric position (J1,2 = 9 Hz). Oacetylated 1-thio-β-D-glucopyranose 1a, N-Ac-1-thio-β-Dglucopyranose 1b, 1-thio-β-D-galactopyranose 1c and 1-thio-β-D-mannopyranose 1d were efficiently coupled with 2 to give corresponding thioglycoconjugates 3a-d. Moreover, the reaction is not limited to monosaccharides, but can be applied to more complex di-and trisaccharide derivatives. Thus, O-acetylated 1thio-β-D-cellobiose 1f and 1-thio-β-D-maltotriose 1g were reacted with 5-iodouridine 2 to give corresponding glycoconjugates 3f and 3g in 55% and 35% yield, respectively. It is noteworthy that fully deprotected sugars can be also used: as an example, the reaction of 2 with 1-thio-β-D-glucose afforded product 3e in satisfactory 46% yield. These results with nucleoside 2 prompted us to explore the coupling of iodine containing oligonucleotides with various thiosugars. 5-iodo-2'-deoxyuridine modification was incorporated into oligonucleotides by using the commercially available C5iodo-dU phosphoramidite and the final deprotection of the nucleobases was achieved in 28% ammonia at room temperature for 24h as recommended (see SI for HPLC profiles and ESMS analysis of iodine containing oligonucleotides). 7-mer ODN T3XT4 (in which X represents 5-iodo-2'-deoxyuridine moiety) and 1-hexanethiol were first chose as model substrates to identify the best conditions for the coupling reaction (Table 1). The reactions were monitored by RP-HPLC and the rate of conversion was estimated from HPLC analysis.

Table 1. Optimization of the coupling reaction of T3-X-T4 with 1-hexanethiol. [a] [a] See the supporting information for the experimental procedure. [b] The rate of conversion was estimated from HPLC profile by comparison of the surface of the peak corresponding to the oligonucleotide conjugate with the surfaces of the other minor peaks.

It was found that the quantity of G3-XantPhos reagent is crucial as the addition of almost 20 equivalents was required to obtain the complete conversion of the starting material (Table 1, entry 4. See also RP-HPLC on Figure A, ESI mass in B and SI: Figure S23 for HPLC profiles). Likewise the quantity of triethylamine must be adjusted to 40 equivalents: addition of less quantity led to incomplete conversion of the starting oligonucleotide (Table 1, entry 4-6, Figure S24 for HPLC profiles). The effect of the temperature was also investigated and revealed a slightly impact on the coupling reaction (data not shown): nevertheless the temperature was kept at 60°C as the room temperature reactions showed some by-products formation. Finally the quantity of 1-hexanethiol must be maintained at almost 10 equivalents. These optimized conditions were applied for conjugation of oligonucleotides T3XAT4, T3XCT4 and T3XGT4 to consider the stability of A, C and G nucleobases during the coupling reaction (Scheme 3). The iodine containing oligonucleotides T3XAT4, T3XCT4 and T3XGT4 were efficiently converted to the corresponding conjugates thus demonstrating that Pd catalyst do not affect the integrity of A, C and G nucleobases (Figures S35-S37 for HPLC profiles). The coupling reaction was then performed with hetero-oligonucleotides TTAGGACAXGCT and GCTXCCACA as well as with the longer T10XT9 oligonucleotide and revealed to be very efficient to afford the corresponding conjugates (Figures S38, S39 and S34, respectively for HPLC profiles). The reaction was also achieved for the incorporation of two reporter moieties by using bismodified oligonucleotide T3XT3XT3. Again, the conversion to the corresponding conjugate was very efficient to afford the bisconjugate (Figure S40 for HPLC profile). All the conjugation products with 1-hexanethiol were purified by reverse-phase HPLC and then characterized by ESMS analysis (Table S2 and Figures S63-S70).

The optimized conditions were then applied for the conjugation of various oligonucleotides sequences with O-acetylated thiocarbohydrates derivatives 1a-g (Scheme 3). The reactions were monitored by RP-HPLC. For all the conjugation reactions, the HPLC profiles of the crude mixtures showed the disappearance of the starting material with the formation of the corresponding thioglyco-oligonucleotide as the major product (Scheme 3B as an example and Figures S41-S54). The rate of conversion for all the conjugation reactions was estimated by HPLC analysis to be superior to 90% except for bis-conjugation of T3XT3XT3 (73%). Then thoroughly purifications including EDTA treatment and centrifugation to separate the palladium containing solid followed by size-exclusion chromatography and next RP-HPLC were performed to remove the traces of Pd (see supporting information page 34). The different corresponding carbohydrate oligonucleotide conjugates were obtained in nearly 80% isolated yields. They were characterized by means of ESMS mass spectrometry analysis that showed a good agreement between the experimentally determined molecular weights and the calculated values (Table S2 and Figures S71-S84). [29] It is noteworthy that the use of unprotected thio-sugars for the coupling reaction was discarded because of purification problems: indeed the RP-HPLC analyses showed quite similar retention times for both starting materials and conjugates. Scheme 3. A/ Palladium-catalyzed thioglycoconjugation of 5-iodo-2'deoxyuridine containing oligonucleotides. Reaction conditions: thiol derivatives (400 nmol) in THF is added to a solution of oligonucleotide (40 nmol), G3-Xantphos Pd (800 nmol), TEA (1600 nmol) in H2O/THF (7/1, v/v) and heated at 60 °C for 20 min. [a] Rate of conversion estimated from HPLC. B/ Example of HPLC profile for the coupling reaction: RP-HPLC chromatogram of crude reaction mixture (on left) of T3XT4 with Glu(OAc)4-SH 1a and corresponding purified conjugate (on right).

In conclusion, we have demonstrated that the palladacycle precatalyst G3-XantPhos displays a high activity for thioglycoconjugation of an array of oligonucleotides. The mild reaction conditions allow direct access to a range of glycoconjugates that are otherwise not easily accessible. We expect this simple and general protocol to be of broad utility for the synthesis and development of new oligoglycoconjugates of interest. In particular, efforts are in progress to incorporate up to four carbohydrate moieties through this strategy to diverse structural DNA architectures such as G-quadruplex or i-motif and to study the recognition of those biomolecular systems with lectins.

Scheme 1 .

 1 Scheme 1. Seminal examples of post functionalization of ODNs through Pdcatalyzed C-C cross-coupling reactions, (8-Br-dG = 8-bromo-2'deoxyguanosine, 5-IdU = 5-iodo-2'-deoxyuridine).

Acknowledgements

Authors gratefully acknowledge CNRS, University Paris-Sud, University Grenoble Alpes, "Agence Nationale de la Recherche" (ANR-15-CE29-0002 and ANR-16-CE11-0006-01) and la Ligue Contre le Cancer through an Equipe Labellisée 2014 for financial support. Biocis laboratory is a member of the

Supporting information for this article is given via a link at the end of the document.

Laboratory of Excellence LERMIT supported by a grant (ANR-10-LABX-33). The NanoBio-ICMG platforms (FR 2607) and Labex ARCANE (ANR-11-LABX-0003-01) are also acknowledged for their support.