Albert Rilliard
email: albert.rilliard@lisn.upsaclay.fr

David Doukhan

Rémi Uro

Simon Devauchelle
email: sdevauchelle@ina.fr

EVOLUTION OF VOICES IN FRENCH AUDIOVISUAL MEDIA ACROSS GENDERS AND AGE IN A DIACHRONIC PERSPECTIVE

Keywords: Gender, Diachrony, Vocal Tract Resonance, Vocal register, Broadcast speech

We present a diachronic acoustic analysis of the voice of 1023 speakers from French media archives. The speakers are spread across 32 categories based on four periods (years 1955/56, 1975/76, 1995/96, 2015/16), four age groups (20-35; 36-50; 51-65, >65), and two genders. The fundamental frequency (F 0 ) and the first four formants (F1-4) were estimated. Procedures used to ensure the quality of these estimations on heterogeneous data are described. From each speaker's F 0 distribution, the base-F 0 value was calculated to estimate the register. Average vocal tract length was estimated from formant frequencies. Base-F 0 and vocal tract length were fit by linear mixed models to evaluate how they may have changed across time periods and genders, corrected for age effects. Results show an effect of the period with a tendency to lower voices, independently of gender. A lowering of pitch is observed with age for female but not male speakers.

INTRODUCTION

Vocal characteristics are an important part of identity, with our voices indicating our gender and other social constructs [START_REF] Sergeant | Gender differences in long-term average spectra of children's singing voices[END_REF]. Voice obviously changes with age, a lot during our development until adulthood [START_REF] Fouquet | Seven and up: individual differences in male voice fundamental frequency emerge before puberty and remain stable throughout adulthood[END_REF], but also later with aging voices [START_REF] Russell | Speaking fundamental frequency changes over time in women: A longitudinal study[END_REF]. Social constructs, and the acoustic characteristics linked to them, may change with cultures -as was described by van Bezooijen for Japanese and Dutch female voice [START_REF] Van Bezooijen | Sociocultural aspects of pitch differences between japanese and dutch women[END_REF]: it is thus essential to study these characteristics in varied cultural and language contexts (and it typically fits this ICPhS theme). Some studies also claimed that voices may have changed across time (for populations of comparable age); this was claimed for female voices, for example in Australia [START_REF] Pemberton | Have women's voices lowered across time? a cross sectional study of australian women's voices[END_REF].

We present here a first acoustic analysis of a corpus of voices extracted from French broadcast archives in a diachronic perspective, trying to balance the selection of speakers according to their age and gender. A total of 1023 speakers were selected, and samples of their vocal production were extracted from the original archive. This corpus aims to extract acoustic cues from a large sample, representative of female and male voices presented in French media, to describe them and their possible changes with time and age. Some studies have described diachronic changes of voices across genders, but these studies are few, may have varying conclusions or population sampling, and address populations from different cultural backgrounds. [START_REF] Pemberton | Have women's voices lowered across time? a cross sectional study of australian women's voices[END_REF] addresses young adult women's voices, with two points in time -while [START_REF] Zou | Diachronic contrastive analysis on read speech in broadcast news: Evidence from pitch and duration[END_REF] studied changes in male voices over five time periods, focusing on media anchors: comparatively few speakers were included (four for the first three periods), with possible longitudinal effects. For French, [START_REF] Suire | Evolution of human pitch: Preliminary analyses in the french population using ina audiovisual archives of vox pops[END_REF] selected short samples of voices of anonymous individuals from media archives (mean duration of 5 s.), comparing F 0 of males and females across 70 years. They report complex patterns of F 0 changes across time, with possible opposite tendencies for genders after the 1960s, but don't control for age, a factor known to induce changes in voice F 0 [START_REF] Stathopoulos | Changes in acoustic characteristics of the voice across the life span: Measures from individuals 4-93 years of age[END_REF]. Vocal characteristics are also affected by vocal effort [START_REF] Berg | The speaking voice in the general population: Normative data and associations to sociodemographic and lifestyle factors[END_REF], which has substantial effects on F 0 [START_REF] Titze | Vocal intensity in speakers and singers[END_REF] and on formants [START_REF] Rilliard | Paradigmatic variation of vowels in expressive speech: Acoustic description and dimensional analysis[END_REF].

Long-term acoustic measures give reliable information on voice characteristics. [START_REF] Löfqvist | Long-time average spectrum of speech and voice analysis[END_REF] showed the long-term average spectrum stabilizes for articulatory changes with about 10 seconds of voiced speech. [START_REF] Arantes | Temporal stability of long term measures of fundamental frequency[END_REF] compared several measures linked to register and found the base-F 0 [START_REF] Traunmüller | The perceptual evaluation of f 0 excursions in speech as evidenced in liveliness estimations[END_REF] was faster to stabilize (with less than ten s. of voiced speech) than mean or median F 0 . A study [START_REF] Johnson | Vocal tract length normalization[END_REF] linked to the estimation of the vocal tract length (VTL) from formants showed the method proposed in [START_REF] Lammert | On shorttime estimation of vocal tract length from formant frequencies[END_REF] was the most rapidly stable, even on a reduced dataset. The literature reports that gender is perceived notably through two acoustic cues -F 0 and supraglottal resonances [START_REF] Pisanski | The prioritization of voice fundamental frequency or formants in listeners' assessments of speaker size, masculinity, and attractiveness[END_REF][START_REF] Leung | Associations between speaking fundamental frequency, vowel formant frequencies, and listener perceptions of speaker gender and vocal femininity-masculinity[END_REF].

The remaining parts of the paper present estimates of F 0 and VTL made on a corpus collected following the semi-automatic strategy proposed by [START_REF] Uro | A semi-automatic approach to create large genderand age-balanced speaker corpora: Usefulness of speaker diarization & identification[END_REF] 1). Some of these categories being much less present in media (typically women), finding target speakers from all age categories in the earliest periods was challenging. It was thus mandatory to increase the targeted period for the 1955-56 and 1975-76 periods by considering the years between 1954-1957 and 1974-1977 (note the additional years only represent a small part of the selected speakers so that these periods will be referred to with the originally targeted years for simplicity). The collection was done thanks to INA's archivists identifying specific women and men within the four age categories and featuring in programs from the four given targeted time periods. Following the method described in [START_REF] Uro | A semi-automatic approach to create large genderand age-balanced speaker corpora: Usefulness of speaker diarization & identification[END_REF], the programs featuring target speakers were submitted to a diarization process, and the ID corresponding to each target speaker was then handpicked. These speakers' samples were then selected to keep samples with minimal noise or background music and remove silences. These archive extracts were submitted to procedures to discard excerpts with adverse characteristics. LIUM_SpkDiarization [START_REF] Meignier | Lium spkdiarization: an open source toolkit for diarization[END_REF] was used to reject segments associated with a telephone quality. Speakers with less than 10 seconds of valid pitch estimates were rejected (the filtering protocol is detailed in section 2.3). Table 1 To estimate robust F 0 and formant measurements from widely different materials (due to heterogeneity in recording and archival conditions), the voicing decision, and then F 0 and formant estimations, were made by concurrent algorithms. The Spleeter source separation framework was used to separate voice from other phenomena (music or noise) [START_REF] Hennequin | Spleeter: a fast and efficient music source separation tool with pre-trained models[END_REF]. The acoustic features were then estimated twice, from both the original and speechseparated signals. For voicing and F 0 estimation, [START_REF] Vaysse | Performance analysis of various fundamental frequency estimation algorithms in the context of pathological speech[END_REF] showed good performances of Praat autocorrelation (ac) algorithm; it also shows that these estimations could be improved in noisy conditions by combining REAPER's voicing estimation with FCN-F0's F 0 estimation (based on neural-network). We thus used Praat [START_REF] Boersma | Praat: doing phonetics by computer (version 6.1.09)[END_REF] for estimating voicing and F 0 (ac algorithm with a 65-650 Hz F 0 range) and for estimating the first four formants (using the recommended settings for female and male voices: 5 formants for a respective ceiling frequency of 5.5 or 5kHz). REAPER algorithm [START_REF] Talkin | Reaper: Robust epoch and pitch estimator[END_REF] was used with two distinct settings (default and Hilbert transform) to obtain two other voicing estimates. FCN-F0 was used with default pitch range (30-1000 Hz), and Viterbi smoothing to estimate F 0 [START_REF] Ardaillon | Fully-Convolutional Network for Pitch Estimation of Speech Signals[END_REF].

Acoustic features filtering

The frames detected as voiced by the six estimations (Praat and REAPER with two sets of parameters on the raw and separated signals) were kept. From these frames, those where the four F 0 estimations (by Praat and FCN-F0 on raw and separated signals) were below a 20% gross error rate threshold were kept. This strategy allowed us to keep 52.2% of the signal's original frames instead of 61.1% obtained with Praat's ac algorithm. From these frames, the formant estimates made on both signals (raw and source-separated) were compared to keep only frames with four formant estimates, with variations below a 20% gross-error-rate threshold (keeping about 96% of the preceding frames) and 50.2% of the signal's original frames.

Long-term features

For each speaker, the valid samples (cf. above section) were grouped in chunks of at least 10s; i.e., for a 36s extract, three chunks of 12s were used. From each of these chunks, two long-term estimates were made: the speaker's base-F 0 (defined as the seventh decile of the F 0 distribution on the chunk [START_REF] Traunmüller | The perceptual evaluation of f 0 excursions in speech as evidenced in liveliness estimations[END_REF][START_REF] Arantes | Temporal stability of long term measures of fundamental frequency[END_REF]), and the speaker's vocal tract length (equal to the median of the length estimations made on each frame based on the first four formants, using the equation proposed in [START_REF] Lammert | On shorttime estimation of vocal tract length from formant frequencies[END_REF][START_REF] Johnson | Vocal tract length normalization[END_REF]). Let's make it clear the estimation of the "vocal tract length" is a way to evaluate the tendency one speaker has to produce higher or lower resonances (formants) as a result of their articulatory habits; the relation to actual vocal tract length is undoubtedly more complex [START_REF] Titze | Toward a consensus on symbolic notation of harmonics, resonances, and formants in vocalization[END_REF]. For each speaker, these two long-term estimates (base-F 0 and vocal tract length: VTL) were estimated for each chunk of voiced samples. The median number of chunks was 14 by speaker, ranging from 1 to 175.

Statistical analysis

The base-F 0 and the estimated VTL were fitted with two linear mixed models that took as fixed effects the actual age (in years) of the speaker, the time period of the recording (four levels: 1955-56, 1975-76, 1995-96, 2015-16), and the speaker's gender -and as random effects the speaker for which the measure was done, and the media program from which the corresponding speaker's chunk was extracted (the program factor was nested in the speaker factor). Following [START_REF] Gries | Statistics for linguistics with R: a practical introduction[END_REF], a maximal model was fit (using R's lme4 library [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF]) that included all the interactions between the fixed factors. This model was then submitted to a simplification procedure to remove non-significant terms and reach a minimal adequate model, one for F 0 and one for VTL. These two models are used here to describe the results of the variation of the base-F 0 and VTL across age, gender, and time period.

RESULTS

Base-F 0

The minimal model fitted on base-F 0 was based on the three main fixed factors (Age, Period and Gender), with the Age:Period and Age:Gender interactions, plus the original random structure (Program nested in Speaker). An estimation of the conditional and marginal coefficients of determination (using [START_REF] Bartoń | MuMIn: Multi-Model Inference[END_REF]) for this model showed the model explained about 90% of the variance, with fixed factors responsible for 58% of this. Figure 1 presents the effect of the Age:Gender interaction on base-F 0 : there is a major difference in F 0 across genders (about 9 st), and F 0 decreased with age for female voices. Figure 2 presents the Age:Period interaction: while Base-F 0 was increasing with Age in the 1950s, it showed a tendency to decrease with age at later periods (1995-96 and 2015-16). Note that this second interaction has a much smaller effect size than the Age:Gender one.

Vocal tract length

The minimal model fitted on VTL was based on the three fixed factors without interaction and keeping the original random structure. The conditional and marginal coefficient of determination for this model showed a much smaller part of the variance was explained by the factors, with the complete model explaining about 38% and fixed factors 31%. The Gender had the largest effect size, with a mean difference of estimated VTL of 1.7 cm between females and males. Period was the next important factor, with a modest but significant increase in VTL of 0.13 and 0.2 cm for the 1995-96 and 2015-16 periods, compared to 1955-56. Age showed a significant but small (0.03 cm for ten years) increasing slope of estimated VTL with age.

DISCUSSION & CONCLUSION

This study intends to bring discussions and gather remarks and advice from the community by presenting preliminary results on a complex topic related to the perception of voice quality, and importantly voice pitch, across gender, age, and time periods in France. As the material used to extract the stimuli could not, for obvious reasons, be of homogeneous quality (recording conditions, signal quality, etc.), there are a series of limitations related to this approach. A problem is linked with the estimation of vocal tract resonances, as highlighted, e.g., by [START_REF] Titze | Toward a consensus on symbolic notation of harmonics, resonances, and formants in vocalization[END_REF]. The quality of archive acoustic quality further complicates this: we had to implement a series of checks to assert we didn't process voices with telephone quality or other deterioration linked to, e.g., compression. The question of the formant estimation algorithm's parameters is essential, as it may lead to varying results. The data presented here are based on the default parameters recommended by Praat for each gender, but this is a potential bias for estimating VTL in speakers with non-standard characteristics. We also tested the parameters recommended by [START_REF] Lammert | On shorttime estimation of vocal tract length from formant frequencies[END_REF] (estimate six formants for a 5.5kHz ceiling frequency): this option led to very different results -and a complex interaction between Gender, Age, and Period with some cases of female speakers having longer estimated VTL than male ones. It was thus not kept here, but questions remain on the estimation of VTL. While distinct settings are generally recommended for the analysis of male and female voices (frequency range for F 0 estimation, frequency ceiling for formats), the use of these a priori settings for the investigation of gender characteristics is questionable since voices, recording, and archival strategies may have changed over the last 60 years -and because it may hide some non-standard features.

Conversely, measures of F 0 seem much more robust and show trends that confirm the literature, with an effect of age on female voice [START_REF] Russell | Speaking fundamental frequency changes over time in women: A longitudinal study[END_REF][START_REF] Yamauchi | Quantitative analysis of digital videokymography: A preliminary study on age-and gender-related difference of vocal fold vibration in normal speakers[END_REF], and a clear gender difference for gender representation in French media. One finding of this study is linked to a change in the evolution of base-F 0 with age across time periods: while it tends to increase in the 1950s for males or stay almost constant for females, steeper slopes were observed in the two later periods (1995-96, and 2015-16). This evolution is not dependent on gender (the interaction was not significant), but the slopes are different for each gender, as shown by figure 2. This decreasing tendency raises questions on the use of voice in public displays (varying social use [START_REF] Boula De Mareüil | A diachronic study of initial stress and other prosodic features in the french news announcer style: corpus-based measurements and perceptual experiments[END_REF] or changing health conditions?), with lowered voices for older speakers across time periods.

Figure 1 :

 1 Figure 1: Fit of base-F 0 (in semitones) from the Speaker Age and Gender.

Figure 2 :

 2 Figure 2: Fit of base-F 0 (semitones) for speaker Age by Period and separated by Gender (males: dashed lines) to show the relative effect of the Period slopes on voices of each gender.

Table 1 :

 1 presents the distribution of the speakers. Voice samples from 1023 speakers were obtained from 878 radio or TV programs (the voice of 85 persons was collected from more than one program). The median duration of valid acoustic features per speaker was 125 seconds. The amount of unique speakers in the 32 categories varies between 15 and 74. Number of speakers per time period (rows), age group, and gender in the corpus 2.2. Voicing, F 0 and Formant estimation

	20-35	36-50	51-65	>65
	F M F M F M F M
	1954-57 17 41 22 74 18 50 17 15
	1974-77 18 17 23 41 28 39 20 26
	1995-96 32 31 32 46 29 47 29 35
	2015-16 30 31 30 52 28 48 29 31

Speech Acoustics

ACKNOWLEDGEMENTS

This work has been funded by the French National Research Agency (project Gender Equality Monitor -ANR-19-CE38-0012). The authors are deeply indebted to INA's archivists Laëtitia Larcher and Anissa-Claire Adgharouamane and who selected the target speakers and also wished to thank Luc Ardaillon and Jean-Hugues Chenot for their valuable advice.


João Antônio De Moraes

Albert Rilliard
email: albert.rilliard@lisn.upsaclay.fr

Macro-and Micro-prosodic Changes in the Duration of Brazilian Portuguese Vowels

Keywords: Vocalic duration, Micro-prosody, Macro-prosody, Brazilian Portuguese

This accounts for the effect of intrinsic segmental characteristics on the duration of Brazilian Portuguese oral vowels, compared to durational changes that can be attributed to macro-prosodic features, such as stress location, sentence modality, and syntactic structure. A multispeakers corpus containing logatomes with systematic variations of vowel quality (seven vowels) embedded in sentences with controlled characteristics was recorded. A semi-automatic procedure allowed to segment and measure the duration of 3036 vowels; their duration was fitted to a series of factors, representing the controlled micro-and macro-prosodic characteristics. This modeling process showed the importance of micro-prosodic changes mainly in the case of stressed vowels (explained by jaw aperture and tongue posteriorization), which decreases with the vocalic reduction on prestress and post-stress positions, depending on macro-prosodic factors. The main macro-prosodic effects are linked to stress, the syllable position, final lengthening, and modality.

INTRODUCTION

Segmental effects on the fundamental frequency (F0), intensity, and duration are referred to as micro-prosodic effects and may influence the description of macro-prosodic changes; thus the choice often made by prosodists to work on lab speech based on controlled sentences. This paper aims to describe quantitatively the variations induced in the duration of Brazilian Portuguese (BP) vowels by both micro-prosodic factors (i.e., the segmental, intrinsic characteristics of these vowels) compared to macro-prosodic ones (stress position in the word, the word's place in the sentence and the sentence modality).

The vocalic system of BP has seven oral vowels, /i e ɛ a ɔ o u/, that form minimal pairs at stress positions, but it reduces to five opposable qualities at pre-tonic positions with the neutralization of the middle vowels /e/ ~ /ɛ/ and /ɔ/ ~ /o/ [1], although the seven qualities are still produced mainly due to vocalic harmony processes [START_REF] Lee | Sobre as vogais pré-tônicas no Português Brasileiro[END_REF]. Post-tonic positions show only three qualities in the word-final position, /i a u/, but may have four or even five qualities (/i e a o u/) in non-final post-tonic syllables (e.g., penultimate syllables of proparoxytonic words) [START_REF] Bisol | Neutralização das átonas[END_REF].

The effect of vowel quality on segmental duration has been evidenced for Dutch or English [START_REF] Nooteboom | Production and perception of vowel duration in Dutch: A Study of Durational Properties of Vowels in Dutch[END_REF], [START_REF] Klatt | Interaction between two factors that influence vowel duration[END_REF], or as part of a larger set of factors (e.g., [START_REF] Van Santen | Contextual effects on vowel duration[END_REF]), as an intrinsic duration factor [START_REF] Cristo | De la microprosodie à l'intonosyntaxe[END_REF], with jaw aperture being the main explication to intrinsic duration changes. Several works have dealt with vocalic duration for Brazilian Portuguese. [START_REF] De Moraes | Durée Intrinsèque des Voyelles du Portugais Brésilien[END_REF] measured the duration of BP vowels of ten speakers from Rio de Janeiro at different sentence positions and confirmed a lengthening linked to aperture until mid-open vowels -/a/ being shorter than /ε/ and /ɔ/; differences along the anteroposterior dimension were observed, but not consistently. Similar results were replicated for another BP dialect, São Paulo [START_REF] Barbosa | Revelar a estrutura rítmica de uma língua construindo máquinas falantes: pela integração de ciência e tecnologia de fala[END_REF]. [START_REF] Escudero | A cross-dialect acoustic description of vowels: Brazilian and European Portuguese[END_REF] observed the duration of BP oral vowels in 20 speakers from São Paulo for stress position of disyllabic paroxytones. Differing from the two previous studies, they showed an increased duration linked to aperture even for the /a/; they also observed some effect on the duration of the front-back articulatory dimension.

The present article targets the duration of BP oral vowels and is part of a wider project aiming at describing micro-prosodic effects (intrinsic and co-intrinsic) in relation to macro-prosodic changes; to that aim, known factors of variation were controlled while creating the corpus. After presenting the corpus construction and recording, the output of the fit of syllable durations to the controlled factors is given and discussed.

METHODS

Corpus construction

The corpus is based on trisyllabic CV1CV2CV3 logatomes (with C being /p/, and Vx on the seven oral vowels of BP /i, e, ε, a, ɔ, o, u/) inserted in the carrier sentence "Ele dirá X [de novo], mas parecerá Y [de novo]" ("He will say X [again], but it will sound like Y [again]"), where X and Y are two different logatomes, and [de novo] an optional extension. The sentence may or not propose an extension of the nominal group containing the logatome, and it was produced with two modalities -as an assertion or an interrogation.

The logatomes had the stress at each possible position (oxytone, paroxytone, proparoxytone). They were constructed with the same vowel at each position (thus, 21 logatomes are considered here: 7 vowels x 3 stress positions), with a limitation of the vowel inventory at unstressed syllables (see introduction).

Recording procedure

Six speakers (3 females and 3 males, mean age 46 years) were recorded in a sound attenuated room, using an omnidirectional measurement DBX RTA PHM919 microphone placed 30cm from their mouth, plugged into a Zoom H5 recorder, with the recording level calibrated by a Brüel & Kjaer 4230 sound level calibrator. Speakers were presented with the sentences in random order via a computer interface and had to read them once -unless the experimenter judged the reading unsatisfactory.

Segmentation and measurements

A forced alignment procedure was applied to the recorded sentences based on their orthographic transcription supplied to the Montreal Forced Aligner [START_REF] Mcauliffe | MontrealCorpusTools/Montreal-Forced-Aligner: Version 2.0.6[END_REF]. Ad hoc phonetization of logatomes was provided to the program. The segmentation was then hand-corrected using Praat [START_REF] Boersma | Praat: doing phonetics by computer[END_REF] by two experienced phoneticians. Considering two sentence modalities, two logatome positions in the sentence, the eventual extension, three vowels per each of 21 logatomes, and six speakers, it ended with 3036 individual vowels. From this dataset, a Praat script extracted the duration of each segment and other measures not presented here.

Statistical analysis

The following factors were controlled: the Logatome position (LogP) -at the syntagm end or not (Boundary or No boundary); the syntagm was at the initial part of the sentence or the end (Syntagm position (SynP): initial or final); the sentences were produced with a given Modality (Mod: Assertive or Interrogative). The logatomes also bear a specific Stress pattern (Stress: Oxitonic, Paroxitonic, Proparoxitonic), and its syllables are at three positions (Syllable position (SylP): Antepenultimate, penultimate, and ultimate). These five controlled factors are linked with macro-prosodic characteristics.

Then, the set of phonological vowels constitutes the micro-prosodic controlled factor (Vowel quality -VowQ). As existing vowels in PB vary according to the syllable position and the word tonal pattern (with syllables bearing the stress or being at pre-or post-stressed positions), different linear mixed models were fitted to the data. A first model was fitted on the cardinal extreme vowels /i a u/, found in all possible levels of the controlled factors (even if reduced at post-stressed positions) to test the effect of all macro-prosodic factors. Then, different mixed linear models were fitted on subsets of syllables, according to their position relative to stress (which determines the number of possible vocalic timbres): one model was made on stressed syllables (7 qualities), one on prestressed vowels (7 qualities with 5 having phonological oppositions), one on post-stress syllables at word-final position (3 qualities), and one on nonfinal post-stress syllables of proparoxytones (5 qualities).

All models were fitted using the same process (following [START_REF] Gries | Statistics for linguistics with R: a practical introduction[END_REF] and using the lme4 library [START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF] of the R software [START_REF] Core | R: A Language and Environment for Statistical Computing[END_REF]): we started by fitting a maximal model, with, as fixed factors, the microprosodic factor (having a varying number of levels as a function of the selected vocalic qualities, from 3 to 7) and the available macroprosodic factors, with all interactions, and a random structure with an intercept for the six speakers (Spk) and for each produced sentence (Snt) nested in the speakers. This maximal model was simplified until reaching a minimal model adequate for describing the change in vocalic duration (Dur, in ms), used as the dependent variable of these models. The following section describes these different models and the main effects observed in each case; ANOVA tables are presented in Tables 1 &2 for the first two models, with the effect size estimated using the partial ω 2 given by the effectsize library [START_REF] Van Santen | Contextual effects on vowel duration[END_REF].

RESULTS

Models of duration

The exact formulas of the minimal models are given in the endnotes for the sake of clarity.

Duration of cardinal vowels

The minimal model 1 was based on all main fixed factors, implied in two four-way interactions linked to macro-prosodic effects (Stress:SylP: SynP:LogP and SylP:SynP:LogP:Mod), and three-way and two-way interactions mixing micro-and macro-prosodic factors (VowQ:SynP and VowQ:Stress:SylP). The random structure kept only the Spk effect. This model explained about 81% of the variance, with fixed factors responsible for 75% (𝑅 𝑚𝑎𝑟𝑔𝑖𝑛𝑎𝑙 2 = 0.75; 𝑅 𝑐𝑜𝑛𝑑𝑖𝑡𝑖𝑜𝑛𝑎𝑙 2 = 0.81). From this complex structure, most of the changes in duration are explained by the Stress:SylP interaction, i.e., if the syllable bears or not the stress. In Figure 1, the durations of stressed vowels are systematically longer than the unstressed ones and above 100ms; most unstressed syllables have a duration between 50 and 75ms. Factors that induce lengthening are syllable position at word end (ultimate) when a syntactic boundary occurs and syntagm at the sentence-final position.

The SylP:SynP:LogP:Mod interaction is essentially linked to a further lengthening of sentence-final vowels (i.e., ultimate syllable of the word, for logatome at syntagm end and syntagm at sentence end), for interrogative modality compared to assertive.

The micro-prosodic effect showed longer /a/ and shorter /i/, with /u/ in between (thus an effect of vowels height and backness dimensions) -but this figure is affected by macro-prosodic characteristics: this holds for the longest vowels (i.e., the stressed ones). Conversely, most unstressed vowels, with centered qualities, showed few to no micro-prosodic differences (see latter models for details).

Duration of stressed vowels

The model on stressed vowels was based on VowQ plus the four-way interaction between SylP, LogP, SynP, and Mod, keeping the complete random structure. There was thus no interaction between the micro-and macroprosodic levels: the VowQ factor, with seven qualities, was replaced by two factors -for the vowel's "Height" (high, mid, low) and "Backness" (front, back)-to reach the minimal model 2 (see The 4-way interaction explains classical macroprosodic lengthening at phrase final boundary, an interaction with modality; details cannot fit here. The two micro-prosodic factors, vowel Height and Backness, show the importance of both characteristics on the duration change, with a relatively important effect size for Heigh (ω 2 =0.2; with a 9ms lengthening from high to mid vowels, and a 12ms one from mid to "low" ones, that groups /a, ε, ɔ/), and reduced effect size for Backness (ω 2 =0.03; with a 9ms lengthening for back vowels over front ones). We did not observe a duration difference between low /a/ and low-mid vowels -thus, a three-level Height factor was sufficient (and /a/ was processed as a front vowel for Backness).

Duration of unstressed vowels

Three models were fitted for the duration of unstressed vowels, which descriptions do not fit here (see formulas in footnotes): one for pretonic vowels 3 , one for final post-tonic vowel 4 , and one for non-final post-tonic 5 . An interaction between micro-and macro-prosodic factors was observed for pretonic vowels, showing that the microprosodic differences no longer apply for macroprosodic factors that most reduce the vocalic duration. This was also observed for non-final post-tonic vowels, the position showing the shortest vowels, where the five vowel qualities have mostly comparable durations. In the case of final post-tonic vowels lengthened because of their position, the three qualities show the same hierarchy described for cardinal vowels (/a/ > /u/ > /i/).

DISCUSSION & CONCLUSION

This work gives a first and rapid analysis of the micro-and macro-prosodic factors that influence oral vowel duration in BP in this dataset. As only intrinsic duration phenomena were considered here, this shall be extended in the future to report on co-intrinsic phenomena and include nasal vowels and diphthongs. The results mostly back what was already described in the literature on micro-and macroprosodic effects. It replicates the same results as [START_REF] De Moraes | Durée Intrinsèque des Voyelles du Portugais Brésilien[END_REF][START_REF] Barbosa | Revelar a estrutura rítmica de uma língua construindo máquinas falantes: pela integração de ciência e tecnologia de fala[END_REF], with /a/ not being the longest vowel but comparable in aperture effect to /ε/ and /ɔ/, a finding that differs from [START_REF] Escudero | A cross-dialect acoustic description of vowels: Brazilian and European Portuguese[END_REF] results (in our data, /ɔ/, not /a/, was the longest stressed vowel: as a "low" -i.e., within the category of longest vowels in our data, for the vowel Height factor-and back vowel). The effect of posterior articulation was systematic in our data for stressed vowels. This differs from the three previous studies, which do not report systematic lengthening with posterior articulation.

Another difference was linked to using a wide set of macro-prosodic factors, the effect of which has been shown here to interact with the microprosodic level. Further investigation may be required to understand when and why this interaction happens and propose an interpretation of the reasons for such behavior, but it seems there is a link between the duration of a vowel and the strength of the micro-prosodic effects, thus they are more obvious on stressed vowels. Vowels with shorter durations tend to be centralized, and this affects the importance of articulatory gestures -thus the reduced microprosodic changes in these cases.

Figure 1 :

 1 Figure 1: mean fitted vocalic duration for the Stress:SylP:SynP:LogP interaction

Table 1 :

 1 Significant factors for the model fit on cardinal vowels. For each factor or interaction: corresponding χ 2 , degree of freedom, p-value, and partial ω 2 . Only significant effects are reported here.

	χ 2	df p	ω 2

Table 2 )

 2 ; the interaction Height: Backness was not significant. The model had a lower (72%) explanatory power, with fixed factors explaining only 37% of the variance: this shows the importance of stress lengthening, which is not accounted for here (considering only stressed vowels), leaving more importance to random factors.

			SynP	Final	Initial	LogP	Boundary	No boundary
			Proparoxitonic				Paroxitonic			Oxitonic
		200
	Vowel duration (ms)	100 150
		50
		Ante.	Penul.	Ultimate		Ante.	Penul.	Ultimate	Ante.	Penul.	Ultimate
						Syllabic position

Table 2 :

 2 Significant factors for the model fit on stressed vowels. For each factor or interaction: corresponding χ 2 , degree of freedom, p-value, and partial ω 2 .

Speech Prosody ID: 632

ACKNOWLEDGMENTS

The authors are deeply grateful to the six speakers that agreed to a considerable effort to record the corpus.


Nicolas Gendron

Mickael Rosa

Adeline Blandinieres

Yoann Sottejeau

Elisa Rossi

Eric Van Belle

Salim Idelcadi

Séverine Lecourt

André Vincentelli

Audrey Cras

Ramadan Jashari

Richard Chocron

Yaël Baudouin

Thibault Pamart

Ivan Bièche

Nathalie Nevo

Bernard Cholley

Jeanne Rancic

Bart Staels

Pascale Gaussem

Annabelle Dupont

Alain Carpentier

Sophie Susen

David M Smadja
email: david.smadja@aphp.fr

Prof M David

Smadja

Aortic valve interstitial cells act as vasculogenic progenitor cells during human calcific aortic valve disease Running title: Gendron et al. Aortic valve interstitial cells and vasculogenesis

Keywords: valvular interstitial cells, calcific aortic valve disease, vasculogenesis, ECFCs, endothelial progenitor, angiogenesis αSMA: α-smooth muscle actin CAVD: calcific aortic valve disease CM: conditioned media ECFCs: endothelial colony forming cells mab VEGFA: monoclonal blocking antibody against VEGF-A MSCs: mesenchymal stem-cells NG2: neural glial antigen-2 VECs: valve endothelial cells VICs: valve interstitial cells VICc: control VIC VICp: pathological VIC

Objective. The study's aim was to analyze the capacity of human valve interstitial cells (VICs) to participate in aortic valve angiogenesis.

Approach and results. VICs were isolated from human aortic valves obtained after surgery for calcific aortic valve disease (CAVD) and from normal aortic valves unsuitable for grafting (control VICs: VICc). We examined VIC in vitro and in vivo potential to differentiate in endothelial and perivascular lineages. VIC paracrine effect was also examined on human endothelial colony forming cells (ECFCs). A pathological VIC (VICp) mesenchymal-like phenotype was confirmed by CD90 + /CD73 + /CD44 + expression and multipotent-like differentiation ability. When VICp were co-cultured with ECFCs, they formed microvessels by differentiating into perivascular cells both in vivo and in vitro. VICp and VICc conditioned media (CM) were compared using serial ELISA regarding quantification of endothelial and/or vasculogenic factors. Higher expression of VEGF-A was observed at the protein level in VICp-CM and confirmed at the mRNA level in VICp compared to VICc.

CM from VICp induced in vitro a significant increase in ECFC proliferation, migration and sprouting compared with CM from VICc. These effects were inhibited by blocking VEGF-A with blocking antibody or siRNA approach, confirming VICp involvement in vasculogenesis by a VEGF-A dependent mechanism.

Conclusions.

We provide here the first proof of a vasculogenic potential of human VICs isolated from CAVD patients. These results point to a novel function of VICp in valve vascularization during CAVD, with a perivascular differentiation ability and a VEGF-A paracrine effect. Targeting perivascular differentiation and/or VEGF-A to slow CAVD progression warrants further investigation.

Introduction

Calcific aortic valve stenosis (CAVD) is a major public health issue and affects 2 to 7% of adults over the age of 65. Its prevalence will double within the next 50 years due to population aging [START_REF] Lindman | Calcific aortic stenosis[END_REF] . CAVD pathogenesis is unclear and is described as a progressive fibro-calcifying process with calcific lesion formation associated with inflammatory cells accumulation, fibrosis and neovascularization of the aortic valve 2,3,4 . There is currently no effective pharmacological treatment to prevent or slow disease progression.

Normal aortic valve displays a layered architectural pattern of cells, including valve endothelial cells (VECs) at the blood-contacting surface and valve interstitial cells (VICs), which are the main cells found in the 3 layers of the valve. VICs are responsible for tissue homeostasis and valve function. They account for a dynamic, heterogeneous cell population, negative for CD31 and CD68, with a fibroblast-like phenotype and the capacity to differentiate into osteoblast-like cells and to promote calcification [START_REF] Rutkovskiy | Valve Interstitial Cells: The Key to Understanding the Pathophysiology of Heart Valve Calcification[END_REF] . A normal valve is avascular and the pathophysiology of aberrant angiogenesis in CAVD is still unknown. We and others have described several mesenchymal-type stem cells able to act as post-natal vasculogenic cells. While bone marrow mesenchymal stem cells (MSCs) [START_REF] Smadja | Angiogenic potential of BM MSCs derived from patients with critical leg ischemia[END_REF] and circulating fibrocytes 7 are able to behave as perivascular cells and to support vasculogenesis via the release of angiogenic factors [START_REF] Smadja | Treprostinil indirectly regulates endothelial colony forming cell angiogenic properties by increasing VEGF-A produced by mesenchymal stem cells[END_REF] , two mesenchymal subtypes isolated from CD133 cells have been described as giving rise to endothelial cells. They are respectively hemangioma stem cells 9,10 isolated from infantile tumors and very small embryonic-like stem cells isolated from human bone marrow 11,12, [START_REF] Smadja | Bone Marrow Very Small Embryonic-Like Stem Cells: New Generation of Autologous Cell Therapy Soon Ready for Prime Time?[END_REF] . Both cell types are able to differentiate into endothelial and perivascular cells.

VICs have been described as mesenchymal cells with a highly plastic phenotype [START_REF] Rutkovskiy | Valve Interstitial Cells: The Key to Understanding the Pathophysiology of Heart Valve Calcification[END_REF] . In 2016, Arevalos et al. showed that porcine VICs exhibited a pericyte-like behavior as they stabilized early VEC vasculogenic networks, expressing α-smooth muscle actin (α-SMA) and neural glial antigen-2 (NG2). However, cellular mechanisms involved in this vasculogenesis process remain unknown, and particularly in the CAVD context.

The aims of this study were 1) to investigate the ability of VICs from CAVD patients to differentiate into endothelial and perivascular lineages and 2), to analyze the paracrine effects of VICs on human endothelial progenitor cells and the subsequent signaling pathways controlling VIC effects on the formation of new vessels in CAVD.

Material and methods

Study population

Study patients were recruited at the cardiovascular surgery department of the CHU in Lille, France. The study protocol, in compliance with the ethical guidelines of the Declaration of Helsinki, was approved by the local ethics committee (ATHERAO study, CPP2008_13/0839). Informed consent was obtained from each patient included. A total of 22 consecutive patients who underwent aortic valve replacement for severe tricuspid CAVD (according to AHA guidelines 14 ) were enrolled.

Human aortic valves were obtained immediately after surgery. As controls, non-calcified human aortic valves were collected from prospective donor hearts considered unsuitable for transplantation by the European Homograft Bank (Clinique Saint-Jean, Brussels, Belgium; n=14) that were rapidly excised and washed in Polyphosphate Buffer Saline (PBS) 1X (Online supplemental Figure 1).

In keeping with the typical characteristics of patients presenting with CAVD, median age of patients was 71.5 years (ranging from 49 to 84) and 63.4% were males (Online supplemental Table 1). Control aortic valves were collected from 14 autopsy hearts from patients with a median age of 52 years (ranging from 32 to 72) with 50% males.

Aortic VIC isolation and culture

Pathological VICs (VICp) and control VICs (VICc) were obtained after type-I collagenase digestion of the aortic valve, as previously described [START_REF] Rosa | Leptin induces osteoblast differentiation of human valvular interstitial cells via the Akt and ERK pathways[END_REF] , from patients after surgery and after autopsy for controls, respectively. VICs were seeded on fibronectin-coated plates (0.4 μg/cm 2 , Millipore, Billerica, MA, USA), cultured in Smooth Muscle Growth Medium-2 (SMGM-2, Lonza, Walkersville, MD, USA) supplemented with 20% FBS (fetal bovine serum, Hyclone, Logan, UT, USA) and used between passages 2-6. The absence of contamination by endothelial cells and/or macrophages was ensured after digestion by flow cytometry with fluorochromeconjugated antibodies CD31 (BD Biosciences, San Jose, CA, USA) and CD68 (BD Biosciences). All assays were performed at 37°C, on 95% sterile air, and 5% CO2 in a saturated humidified incubator.

ECFC and MSC isolation and culture

Human umbilical cord blood samples from healthy, full-term newborns were obtained through a partnership with the Cord Blood Bank of St Louis Hospital (Paris, France), which is authorized by the French Regulatory Authority (authorization no. PPC51) and participates in scientific research. Cord blood endothelial progenitor cells (ECFCs) were isolated from the adherent mononuclear cells fraction as previously described [START_REF] Melero-Martin | Chapter 13. An in vivo experimental model for postnatal vasculogenesis[END_REF][START_REF] Smadja | Bone morphogenetic proteins 2 and 4 are selectively expressed by late outgrowth endothelial progenitor cells and promote neoangiogenesis[END_REF][START_REF] Smadja | Standardization of methods to quantify and culture endothelial colony-forming cells derived from peripheral blood: Position paper from the International Society on Thrombosis and Haemostasis SSC[END_REF] . ECFCs were expanded on fibronectin-coated plates using Endothelial Growth Medium-2 (EGM-2, Lonza) supplemented with 5% FBS. MSCs were isolated from the mononuclear cell fraction of human adult bone marrow as previously described and according to International Society for Cellular Therapy standard criteria for MSC isolation and characterization [START_REF] Dominici | Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement[END_REF] . The medium was replaced every 2 or 3 days until confluence. Angiogenic assays have been described elsewhere 12,15,20 and are detailed in the supplementary file.

Statistical analysis

Data are shown as mean ± SEM. Assessment of parametric model assumptions was performed using Levene's test of equal variances and Shapiro's test of normal distribution. Data sets failed normality and equal variance tests and were analyzed using the Mann-Whitney nonparametric test or Kruskal-Wallis test followed by the Dunn multiple comparisons test. All statistical analyses were performed using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA) and the StatView software package (SAS, Cary, NC, USA).

Differences were considered significant at p<0.05 and shown as * p<0.05, **, p<0.01 and ***, p <0.001.

Results

VICs isolated from CAVD patients exhibit a mesenchymal phenotype

Human VICp in monolayer cultures showed the elongated, spindle-shaped morphology characteristic of mesenchymal cells in culture 21 (Figure 1A). ECFCs served as positive controls for endothelial and vasculogenic cells. VICp were found negative for endothelial (CD144, Tie-2 and CD31) and progenitor markers (CD133 and CD34), positive for CD90, CD73 and CD44 at the same level as MSCs (Figure 1B) and positive for integrins αv, β1 and β3. Furthermore, mesenchymal, endothelial and hematopoietic marker gene expression was quantified by RT-qPCR (Figure 1C). Cultured VICp expressed a high level of mesenchymal markers such as THY1 (CD90), CD44 and fibroblast markers such as VIM, S100A4 and TWIST1, at similar levels as MSCs. Moreover, VICp expressed low levels of endothelial markers such as FLT1 and KDR, contrary to ECFCs. All isolated and cultured MSCs, VICp and ECFCs expressed extremely low levels of hematopoietic markers such as PTPRC (CD45) and fat cell markers such as LEP and ADIPOQ. We then explored the multipotent differentiation ability of VICp. In an osteogenic medium, VICp underwent osteogenic differentiation firstly revealed by ALP activity and then by calcium deposits as detected by Alizarin Red staining (Figure 1D). Human VICp were cultured on fibronectin-coated tissue culture plastic because our objectives were to explore the angiogenic properties and vasculogenic differentiation of VICp. Calcification assays were evaluated in different matrix-coated tissue conditions in the absence of coating, gelatin or fibronectin (Online supplemental Figure 2). When VICp were cultured in control or osteogenic cell differentiation medium, no significant differences in Alizarin Red staining were observed between the different matrices evaluated (p>0.05 for each). We also explored calcium deposition by VICp cultured alone or with ECFCs in control medium or in osteogenic cell differentiation medium (Online supplemental Figure 3). No significant differences in Alizarin Red staining were observed between VICp alone or cocultured with ECFCs (p>0.05 for each).

VICs from CAVD patients differentiate into perivascular cells but not into endothelial lineages

In order to assess whether human VICp were able to differentiate into endothelial and/or perivascular lineages, VICp alone, ECFCs alone, or VICp+ECFCs were suspended in Matrigel and injected into nude mice as previously described 12,22 (Figure 2A). Control matrigels used MSCs+ECFCs. After 10 days, explanted Matrigel plug sections showed numerous vascular channels filled with red blood cells in the Matrigel explants containing VICp+ECFCs, contrary to those containing VICp or ECFCs alone, that did not form vessels (Figure 2B). Interestingly, matrigel explants containing VICp+ECFCs showed similar microvessel density (0.012 ±0.002 vessels/µm²) than control explants MSCs+ECFCs (0.017 ±0.003 vessels/µm², p=0.23, Figure 2B, right panel).

In these explants, immunofluorescence analysis for SMA and human-specific CD31 showed perivascular cells positive for SMA surrounding human CD31 blood vessels. On the contrary, explanted Matrigel containing ECFCs alone contained CD31-positive cells but no αSMA-positive cells. These results suggest that no αSMApositive cells migrated from the mouse into the matrigel plugs during the experiment.

Matrigel explants with VICp alone did not show either any vascularization (Figure 2C) in contrast to what we previously observed with hemangioma stem cells 10 or with very small embryonic-like stem cells 11 . To confirm this finding, we investigated the ability of VICp to differentiate in vitro into endothelial lineages under culture in the presence of VEGF-B, as previously described 11,23 . After 14 days, no induction of VE-cadherin expression was found, thus confirming in vivo data (Online supplemental Figure 4).

The ability of VICp to differentiate into perivascular SMA cells was then explored in vitro with coculture experiments. In order to investigate expression changes in perivascular markers in coculture 50:50, we investigated the expression of perivascular markers on VICp alone and after coculture with ECFCs (Figure 3A).

After confirmation that ECFCs did not express any significant level of the pericyte marker NG2 (Online supplemental Figure 5), we cocultured VICp for 10 days with ECFCs and demonstrated a significant increase in the mRNA expression of NG2 compared to ECFCs alone (Figure 3B). Moreover, VICp acquired a perivascular phenotype confirmed by the induction of SMA, NG2 and calponin protein in confocal analysis (Figure 3C).

VICp without direct contact with ECFCs did not express higher levels of these proteins after 10 days as evaluated by fluorescence intensity quantification (Online supplemental Figure 6). Because ECFCs are known to be involved in perivascular differentiation through paracrine PDGF-BB signaling [START_REF] Lin | Human endothelial colony-forming cells serve as trophic mediators for mesenchymal stem cell engraftment via paracrine signaling[END_REF] , we investigated PDGF-BB involvement in VICP/ECFCs coculture to explore its potential modulation in VIC differentiation into perivascular cells. We did not observe any significant difference in PDGFB mRNA levels between ECFCs alone and ECFCs cocultured with VICp for 5 days (Online supplemental Figure 7).

VEGF-A upregulation in pathologic VICs increases vasculogenesis

Given that a normal valve is avascular and becomes vascularized in CAVD 2, [START_REF] Mazzone | Neoangiogenesis, T-lymphocyte infiltration, and heat shock protein-60 are biological hallmarks of an immunomediated inflammatory process in end-stage calcified aortic valve stenosis[END_REF] , we compared the vasculogenic effects of conditioned media (CM) from VICc isolated from normal aortic valves unsuitable for grafting and VICp. First, CM from VICp induced in vitro a significant increase in ECFC proliferation compared to CM from VICc (Figure 4A). Second, we evaluated the effects of CM from VICs on ECFC migration in a modified Boyden chamber assay that consisted in quantifying the cells bound on the outer membrane surface. CM from VICp induced a significant increase in ECFC migration compared to CM from VICc (Figure 4B, Boyden chamber).

Then, to evaluate the ability of VICp to induce ECFC sprouting, we used a three-dimensional fibrin gel assay model with ECFCs seeded onto Cytodex® 3 microcarrier beads and embedded in fibrin gel (Figure 4C). This model is classically used with a fibroblastic feeder on top of the fibrin gel [START_REF] Nakatsu | Optimized fibrin gel bead assay for the study of angiogenesis[END_REF] . For the present experiment, MSCs, VICp and VICc were used as feeders to induce endothelial sprouting, evaluated by the capillary length and the number of branches per bead. In vitro, ECFC sprouting in fibrin gel was stimulated by VICp and MSCs, but not by VICc (Figure 4D, E and F), confirming the proangiogenic phenotype of VICp isolated from calcified valves.

To further investigate the proangiogenic paracrine effects of CM from VICs, levels of angiogenic factors were screened (Table 1). CM from both VICc and VICp contained similar amounts of Ang-1, Ang-2, sVEGFR-1, PlGF, endothelin-1, sEPCR, sENG, TSP1 and IL-6. No detectable amounts of sVEGFR-2 and VEGF-B were found. Interestingly, a 15-fold higher VEGF-A level was found in CM from VICp compared to VICc (Table 1), further confirmed at the mRNA level (Figure 5A). In order to assess whether VEGF-A plays a role in VICp angiogenic functions and in adult postnatal vasculogenesis, an inhibition strategy targeting VEGF-A expression in VICp was set up using monoclonal blocking antibodies (mAb VEGF-A) and RNA interference (siVEGFA).

ECFCs were exposed for 72h to CM from VICp incubated with and without mAb VEGF-A. A significant decrease in ECFC proliferation and migration was reported when CM from VICp were preincubated with blocking mAb VEGF-A (Figure 5B and 5C, p<0.05). We further used CM from VICp transfected with specific VEGFA siRNA that generated 80% gene expression inhibition 2 days after transfection (Online supplemental Figure 8). CM were collected from VICs silenced for VEGFA or transfected with scrambled-siRNA and incubated for 3 days with ECFCs. A significant decrease in ECFC proliferation and migration was observed with CM from VICp transfected with siVEGFA, confirming VEGF-A involvement in VICp-induced endothelial proliferation and migration (Figure 5D and5E). Furthermore, using the three-dimensional fibrin gel assay model with ECFCs seeded onto Cytodex® 3 microcarrier beads, VEGFA inhibition with siRNA in VICp used as a feeder significantly decreased ECFC sprouting network formation (Figure 6A, 6B and 6C). Taken together, these results indicate that VEGF-A is overexpressed in VICp from CAVD patients and promotes vasculogenesis.

VEGF-A modulation in VICp does not alter their osteogenic or perivascular differentiation

Firstly, we investigated VEGF-A involvement in VICp osteogenic and perivascular differentiation. Calcification assays were performed in the presence of mAb VEGF-A. No significant differences in Alizarin Red staining were observed when VICp were cultured in the presence or absence of mAb VEGF-A (p>0.05 for each, Figure 7A). RUNX2 has been described as driving VICp osteogenic potential during CAVD [START_REF] Towler | Oxidation, inflammation, and aortic valve calcification peroxide paves an osteogenic path[END_REF] and VEGFA gene expression [START_REF] Kwon | Physical and functional interactions between Runx2 and HIF-1α induce vascular endothelial growth factor gene expression[END_REF] . We explored whether RUNX2 could modulate VEGFA expression in VICp and found that suppressing RUNX2 with siRNA did not modify VEGFA mRNA expression at any time (p>0.05 for each, Online supplemental 9). We then investigated whether mAb VEGF-A could interfere with perivascular differentiation during VICp+ECFCs coculture (Figure 7B) and we did not observe any significant difference between SMA and calponin expression (p>0.05 for each condition, Figure 7C andD). Cellular mechanisms involved in vasculogenesis process associated with CAVD remain unknown. After analysis of VEGFA gene promoter, we identified putative binding sites for HIF1a, LXRA:RXRA and SREBP (Figure 8A). At the mRNA level, VICp significantly overexpressed HIF1a compared to VICc (p=0.0159, Figure 8B) whereas there was no statistical differences in LXRA:RXRA nor in SREBP expression (Figure 8C,D,E). Interestingly, a significant correlation existed between HIF1a mRNA and VEGFA mRNA gene expression in VICs (p=0.0006, r²=0.9119, Figure 8F).

Discussion

We show that VICp isolated from CAVD patients are vasculogenic cells, given their ability to differentiate into perivascular cells and to secrete VEGF-A. To our knowledge, we provide here the first exploration of the vasculogenic potential of VICs whether they were isolated from pathological or control human valves. In CAVD, VICp showed an increased ability to promote vasculogenesis by a paracrine effect related to a burst of VEGF-A secretion. Indeed, inhibition of VEGF-A in VICp through mAb or siRNA blocking strategies decreased their vasculogenic ability. This suggests that VEGF-A could directly contribute to newly-formed aortic valve vessels in CAVD.

Two studies previously proposed this triple association between VEGF-A, angiogenesis and CAVD. First, Rajamannan et al. observed that valve calcification was not a random passive process. Thus, using immunohistochemistry, they found that osteoblasts in calcified rheumatic valves were associated with proliferating myofibroblasts and neoangiogenic markers such as VEGF-A 2 . Second, by isolating mast cells and myofibroblasts from CAVD, Syväranta et al. demonstrated that both cells could secrete VEGF-A, in particular myofibroblasts when cultured with mast cells on hypoxia, inflammation and tobacco 4 . Furthermore, Porras et al. used biomaterial environment models of CAVD [START_REF] Porras | Creation of disease-inspired biomaterial environments to mimic pathological events in early calcific aortic valve disease[END_REF] based on extracellular matrix variation to show that glycosaminoglycan modifications, in particular increasing hyaluronic acid, induced major secretion of VEGF-A by resident VICs but not PDGF or inflammatory cytokines. Moreover, global changes of the miRNA expression profile in CAVD and controls were recently identified, including miR-21-5p microRNA, which is potentially involved in angiogenic and VEGF-related disorders 34,35,36 . Our findings provide evidence that blocking VEGF-A could have a pivotal role in maintaining normal valve functions without interfering with osteogenic potential. However further investigations are needed to find appropriate strategies to block VEGF-A secretion in VIC during CAVD.

Mainly using in vitro models, VICs have been shown to be progenitor cells with major plasticity properties 5 .

Recently, Arevalos et al. showed a pericyte-like behavior of porcine VIC in stabilizing early VEC vasculogenic networks with α-SMA and NG2 expression [START_REF] Arevalos | Valve Interstitial Cells Act in a Pericyte Manner Promoting Angiogensis and Invasion by Valve Endothelial Cells[END_REF] . Herein we demonstrate for the first time in human aortic valves that VICs cultured with endothelial cells can undergo perivascular differentiation and may stabilize aberrant neoangiogenesis during CAVD. The present study also reveals the incapacity of human VICp isolated from CAVD to differentiate into endothelial cells, in contrast to other plastic mesenchymal subtypes. MSCs have been shown to support angiogenesis 6 through the release of angiogenic factors, in particular VEGF-A 8 , and enhance angiogenesis by differentiation into perivascular cells but are incapable of endothelial differentiation [START_REF] Melero-Martin | Engineering robust and functional vascular networks in vivo with human adult and cord bloodderived progenitor cells[END_REF] .

In the present study, we demonstrated that VICs are CD34 -CD133 -and show an mRNA profile comparable to MSCs and/or fibrocytes. VICp are not able to differentiate into cells with the endothelial phenotype in vivo or when cultured with VEGF-B in vitro. However, we cannot exclude a potential endothelial differentiation by other pathways. According to these results, absence of endothelial differentiation excludes VICs them from stem cells and makes them a valve progenitor already committed in non-endothelial lineages. Specific contribution of these cells to valve plasticity during both homeostasis and response to injury is incompletely understood. As Most research on CAVD is drawn from animal cells (porcine or sheep). The strength of our study is to have studied isolated and cultured human cells from calcified tricuspid aortic valves and non-calcific valves collected from prospective donor hearts considered as high-quality control material [START_REF] Rutkovskiy | Valve Interstitial Cells: The Key to Understanding the Pathophysiology of Heart Valve Calcification[END_REF] . Some limitations of this study should be mentioned. First, all the CAVD patients included presented the severe form of the disease, aortic valve stenosis, with heavily calcified leaflets. Our findings may therefore not be representative of the earlier processes of CAVD. Second, we used ECFCs as endothelial cells for all the experiment and not aortic VECs covering valve leaflets. ECFCs are progenitor cells committed in endothelial lineage. In CAVD, ECFCs from bone marrow may be mobilized and recruited to participate in VEC regeneration [START_REF] Matsumoto | Reduced number and function of endothelial progenitor cells in patients with aortic valve stenosis: a novel concept for valvular endothelial cell repair[END_REF] and/or aberrant angiogenesis related to VEGF-A. We found that ECFCs induced VIC-perivascular differentiation but further study are warranted to confirm this interaction with mature endothelial cells (or VECs). Furthermore, VEGF-A secretion may be responsible of one of the early events in CAVD: disruption of the endothelial layer on the aortic side of the leaflets 48 . VECs have not been used in this study since they have been described as giving rise to VICs in the pulmonary and mitral valves 49, [START_REF] Wylie-Sears | Mitral valve endothelial cells with osteogenic differentiation potential[END_REF] . Since we wanted to study the differentiation potential of VICs in CAVD, we did not want to induce any bias with VEC-differentiation interference in these pathways. Lastly, it would be interesting to correlate plasma VEGF-A levels in patients with CAVD and valve function, calcification and angiogenesis. Indeed, immunohistochemical studies of explanted stenotic aortic valves showed a correlation between VEGF-A levels and neovessels in calcified areas 4, [START_REF] Perrotta | HIF-1α and VEGF: Immunohistochemical Profile and Possible Function in Human Aortic Valve Stenosis[END_REF] . In the present study, we observed in VICs a significant correlation between VEGFA and HIF1a expressions. As HIF1a shows putative binding sites for VEGFA gene promoter, further study should explore the interaction between HIFa and VEGFA during CAVD onset. As normal aortic valves are avascular and non-calcific valves are rarely-used control materials, we acknowledge that vasculogenic experiments with VICc were not performed. VICc enabled us to identify changes in the paracrine secretion of VICp in CAVD. These control valves are clearly validated as a high-quality control material [START_REF] Rutkovskiy | Valve Interstitial Cells: The Key to Understanding the Pathophysiology of Heart Valve Calcification[END_REF] and this is a strong argument to validate our results. Moreover, results from these experiments were validated in VICp since VEGF-A inhibition was clearly associated to a modified phenotype and abolished functional differences between VICp and VICc. XXXX rajouter un mot sur l'absence de vrai modèle in vivo

Conclusion

Our study demonstrates that VICp isolated from CAVD patients have higher vasculogenic potential compared to controls and that angiogenesis in CAVD is an active process driven by the native cells in the aortic valve through VIC differentiation into perivascular cells. We highlight the perivascular potential of human VICs and their ability to modify their secretome during disease through increased VEGF-A secretion. Further studies are needed to decipher the clinical relevance of the vasculogenic properties of VICs during CAVD development.

Tables

  mentioned by Liu et al., VICs have a progenitor phenotype 38 , especially during valve formation in the embryo and injured/diseased valves. Additionally, c-Kit + progenitor cells have been observed in healthy and pathological valves 39 , and bone marrow-derived stem cells migrate to human and mouse cardiac valves and differentiate into fibroblast-like cells 40,41 . Our results are in agreement with the hypothesis that VICs are bone marrowmesenchymal derived cell valve residents 40,43,44 . Other progenitor cells isolated from the heart are involved in cardiac repair by enhancing angiogenesis in a paracrine manner, such as c-Kit + cardiac stem cells and MSCs 44,45,46 .

Figure 1 .

 1 Figure 1. Mesenchymal-like phenotype of pathological VICs.

Figure 2 .

 2 Figure 2. Pathological VICs are able to differentiate into perivascular cells.A) MSCs and VICp with ECFCs (1.5×10 6 of each cell, 1:1) were co-injected and 3×10[START_REF] Smadja | Angiogenic potential of BM MSCs derived from patients with critical leg ischemia[END_REF] VICp were injected into

Figure 3 .

 3 Figure 3. Pathological VICs (VICp) differentiate into perivascular cells when cocultured with cord blood

Figure 4 .

 4 Figure 4. Angiogenic potential of VICp on ECFCs.

Figure 5 .

 5 Figure 5. Blocking mAb and siRNA against VEGF-A abolished VICp-induced ECFC proliferation and

Figure 6 .

 6 Figure 6. Silencing VEGFA abolished VICp-induced ECFC sprouting.

Table 1 . VICp show increased VEGF-A secretion compared to VICc.

 1 Conditioned media from VICp contained a significant higher level of VEGF-A compared to VICc. Results are mean ± SD. n=4 to 6 independent experiments each condition performed in triplicate. Statistical tests used:

	Mann-Whitney test.
				VICc vs VICp
	Protein	VICc	VICp
				(p-value)
	Angiopoietin-1 -pg/10 5 cells	68.42 ±124.7	175.0 ±106.3	p=0.065
	Angiopoietin-2 -pg/10 5 cells	411.1 ±495.8	116.6 ±118.4	p=0.32
	VEGF-A -pg/10 5 cells	37.59 ±22.3	575.0 ±83.0	p=0.008
	VEGF-B -pg/10 5 cells	0	0	NA
	PlGF -pg/10 3 cells	2.88±2.8	0.90±1.2	p=0.40
	IL-6 -ng/10 5 cells	0.34 ±0.5	6.55 ±14.8	p=0.25
	Thrombospondin-1 -ng/10 5 cells	26.0±24.4	43.9±26.9	p=0.39
	soluble VEGFR-1 -pg/10 5 cells	4.02±4.2	8.89±5.1	p=0.25
	soluble VEGFR-2 -pg/10 5 cells	0.0	0.0	NA
	soluble EPCR -ng/10 5 cells	48.2±28.37	63.0±51.5	p=0.88
	Endothelin-1 -pg/10 5 cells	1.18±0.8	0.51±0.2	p=0.57
	soluble endoglin -ng/10 5 cells	0.29±0.3	0.16±0.1	p=1.0

Acknowledgments

We would like to thank the Animal Platform, CRP2 -UMS 3612 CNRS -US25 Inserm-IRD -Faculté de Pharmacie de Paris, Université de Paris, Paris, France. We would also like to thank Bruno Saubamea and Virginie Mignon from the Cellular and Molecular Imaging Facility, INSERM US25/CNRS UMS 3612-Faculté de Pharmacie de Paris, Université de Paris, Paris, France. We are indebted to the nursing services at Hôpital des Diaconesses (Paris, France) and Begin (Saint Mandé, France) for providing umbilical cord blood samples.

A) Promoter analysis performed using ConTra v3 web server to predict transcription factor binding sites of VEGFA promoter. ConTra v3 web server identifies putative binding sites for HIF1a, LXRA:RXRA and SREBP. B) Using real-time RT-qPCR, a significant increased mRNA relative expression of HIF1A is found in VICp in contrast to VICc. Results represent the mean ± SEM of 5 independent experiments using VICp and VICc from different donors. * and *** for significant differences with p<0.05. Statistical tests used: Mann-Whitney test. C) Using real-time RT-qPCR, no significant difference in mRNA relative expression of SREBP exists between VICp and VICc (p>0.05). Results represent the mean ± SEM of 5 independent experiments using VICp and VICc from different donors. Statistical tests used: Mann-Whitney test. D) Using real-time RT-qPCR, no significant difference in mRNA relative expression of LXRA exists between VICp and VICc (p>0.05) Results represent the mean ± SEM of 5 independent experiments using VICp and VICc from different donors. Statistical tests used: Mann-Whitney test E) Using real-time RT-qPCR, no significant difference in mRNA relative expression of RXRA exists between VICp and VICc (p>0.05). Results represent the mean ± SEM of 5 independent experiments using VICp and VICc from different donors. F) Correlation between mRNA relative expression of HIF1A and mRNA relative expression VEGF-A in calcified areas of aortic valves (p=0.0006, r²=0.9119). Results represent the mean ± SEM of 5 independent experiments using VICp and VICc from different donors. Statistical tests used: Pearson's correlation coefficient.

Sources of Funding

Disclosures

The authors do not have any conflicts of interest to declare.

Highlights:

 Human VICs from calcified aortic valves gain increased VEGF-A secretion potential allowing ECFC migration, proliferation and sprouting.

 Human VICs from calcified aortic valves are able to differentiate in vivo and in vitro into perivascular cells, independently from VEGF-A.

 Blocking VIC vasculogenic potential could be an innovative pharmacological approach to prevent or slow CAVD progression.