Rana Zahreddine

Morgane Davezac

Natalia Smirnova

Melissa Buscato

Emeline Lhuillier

Adrien Lupieri

Romain Solinhac

Alexia Vinel

Emilie Vessieres

Daniel Henrion

Emeline Lhullier

Marie-Ange Renault

Alain-Pierre Gadeau

Gilles Flouriot

Françoise Lenfant

Muriel Laffargue

Raphaël Métivier

Jean- François Arnal

Coralie Fontaine

Dr Jean-François Arnal
email: jean-francois.arnal@inserm.fr

Tamoxifen Accelerates Endothelial Healing by Targeting ERα in Smooth Muscle Cells

Keywords: Estrogen, tamoxifen, estrogen receptor (ERα), vascular endothelium, smooth muscle cell, endovascular repair. Nonstandard Abbreviations and Acronyms: AF1: Activation Function-1 AF2: Activation Function-2 AI: Aromatase Inhibitors CVD: Cardiovascular Diseases E2: 17β-Estradiol ECs: Endothelial cells

INTRODUCTION

Cardiovascular diseases (CVD) have become an increasingly important cause of long-term morbidity and mortality among breast cancer survivors, mainly because of the improvement of survival after the disease and the increase in the duration of life expectancy. The closed link between breast cancer and CVD, was recently reviewed [START_REF] Laxmi | Cardiovascular Disease and Breast Cancer: Where These Entities Intersect: A Scientific Statement From the American Heart Association[END_REF] , demonstrating shared risk factors and cardiovascular toxicity effects of cancer therapy. The treatments of patients with estrogen receptor α (ERα+) breast cancer are mainly endocrine therapies such as tamoxifen (TAM) and/or aromatase inhibitors (AIs) that are both very efficient in reducing the risk of cancer recurrence in women 2 but also in men [START_REF] Fentiman | The endocrinology of male breast cancer[END_REF] . However, AIs appears to increase the risk of CVD through suppression of the cardiovascular protective effects of estrogens [START_REF] Meyer | Estrogens and Coronary Artery Disease: New Clinical Perspectives[END_REF][START_REF] Matthews | Long term adjuvant endocrine therapy and risk of cardiovascular disease in female breast cancer survivors: systematic review[END_REF][START_REF] Matthews | cancer survivors: two cohort studies in the UK and US[END_REF] , whereas TAM seems to prevent acute myocardial infarction [START_REF] Matthews | Long term adjuvant endocrine therapy and risk of cardiovascular disease in female breast cancer survivors: systematic review[END_REF][START_REF] Matthews | cancer survivors: two cohort studies in the UK and US[END_REF] . Experimental data in mice directly demonstrated several vasculoprotective effects of TAM. Indeed, TAM prevents atherosclerosis [START_REF] Jill | Tamoxifen Decreases Cholesterol Sevenfold and Abolishes Lipid Lesion Development in Apolipoprotein E Knockout Mice[END_REF] and neointimal hyperplasia after artery injury [START_REF] Smirnova | The Activation Function-1 of Estrogen Receptor Alpha Prevents Arterial Neointima Development Through a Direct Effect on Smooth Muscle Cells[END_REF] . These beneficial vascular effects are mediated by ERα [START_REF] Smirnova | The Activation Function-1 of Estrogen Receptor Alpha Prevents Arterial Neointima Development Through a Direct Effect on Smooth Muscle Cells[END_REF][START_REF] Fontaine | Tamoxifen elicits atheroprotection through estrogen receptor α AF-1 but does not accelerate reendothelialization[END_REF] .

ERα belongs to the nuclear receptor family and can regulate gene transcription through activation functions (AF)-1 and/or (AF)-2. Whereas E2 activates both ERα AFs, TAM exerts antagonist actions on ERαAF2 and agonist actions on ERαAF1 [START_REF] Smirnova | The Activation Function-1 of Estrogen Receptor Alpha Prevents Arterial Neointima Development Through a Direct Effect on Smooth Muscle Cells[END_REF][START_REF] Fontaine | Tamoxifen elicits atheroprotection through estrogen receptor α AF-1 but does not accelerate reendothelialization[END_REF][START_REF] Feng | Nuclear Receptor Modulation -Role of Coregulators in Selective Estrogen Receptor Modulator (SERM) Actions[END_REF][START_REF] Shang | Molecular determinants for the tissue specificity of SERMs[END_REF] . In addition to these classical, nuclear, genomic actions, a sub-population of ERα is present at/or near the plasma membrane where it can elicit rapid, non-genomic, membrane-initiated steroid signaling (MISS) effects [START_REF] Arnal | Membrane and Nuclear Estrogen Receptor Alpha Actions: From Tissue Specificity to Medical Implications[END_REF] . We previously demonstrated that 17β-estradiol (E2) accelerates endothelial healing after electric injury through ERα, but not through ERβ [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF] , and more precisely through membrane ERα [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF] actions in endothelial cells (ECs) [START_REF] Toutain | Estrogen receptor alpha expression in both endothelium and hematopoietic cells is required for the accelerative effect of estradiol on reendothelialization[END_REF] . The endothelium has numerous functions, acting as a crucial barrier between the blood and the vessel and regulating vascular tone [START_REF] Krüger-Genge | Vascular Endothelial Cell Biology: An Update[END_REF] . In contrast, abnormalities of endothelial function and loss of the integrity of the endothelial monolayer constitute the key step in the onset of atherosclerosis [START_REF] Gimbrone | Endothelial Cell Dysfunction and the Pathobiology of Atherosclerosis[END_REF] . Furthermore, massive endothelial death/erosion is directly responsible for thrombus formation and cardiovascular events in about one-third of the cases of acute coronary syndromes [START_REF] Franck | Haemodynamic stress-induced breaches of the arterial intima trigger inflammation and drive atherogenesis[END_REF][START_REF] Libby | Atherosclerosis[END_REF] . The therapeutic treatment of both chronic and acute arterial diseases that includes angioplasty followed by stent implantation destroys the endothelium. Finally, radiotherapy and chemotherapies used in cancer treatment also concur to alter endothelium integrity [START_REF] Baselet | Pathological effects of ionizing radiation : endothelial activation and dysfunction[END_REF][START_REF] Soultati | Endothelial vascular toxicity from chemotherapeutic agents: preclinical evidence and clinical implications[END_REF] . In all these interventions, delayed endothelial healing represents one of the important contributing factors of thrombosis and neointimal hyperplasia [START_REF] Cornelissen | The effects of stenting on coronary endothelium from a molecular biological view: Time for improvement?[END_REF] .

In the present study, we then sought to delineate the arterial protection conferred by TAM in three complementary mouse models of arterial injury, i.e. in vivo and ex vivo model of endovascular injury with preserved smooth muscle layers and in vivo model of electric injury in which both endothelium and media t 4 are destroyed. Cre-Lox models were further used to distinguish between the endothelial and the smooth muscle cells (SMCs) specific actions of TAM and E2. The molecular mechanisms underlying the effects of TAM were finally characterized using transgenic mice in which the ERαAF1 and the membrane ERα functions have been specifically invalidated.

METHODS

Materials and data that support the findings of this study are available from the corresponding authors upon reasonable request. For research materials listed in the Methods and Materials please see the Major Resources Table in the Online Supplemental Materials.

Mice.

All procedures involving experimental animals were performed in accordance with the principles and guidelines established by the National Institute of Medical Research (INSERM) and were approved by the local Animal Care and Use Committee. The investigation conforms to the directive 2010/63/EU of the European parliament. Wild-type female mice with a C57Bl/6J background were purchased from Charles River Laboratories (France), (n=4-12 per group). Tie2CreERα lox/lox , αSMACreER T2 ERα lox/lox , ERαAF1 0 and ERα-C451A mouse line were generated as described previously [START_REF] Smirnova | The Activation Function-1 of Estrogen Receptor Alpha Prevents Arterial Neointima Development Through a Direct Effect on Smooth Muscle Cells[END_REF][START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF][START_REF] Billon-Galés | Endothelial estrogen receptor-alpha plays a crucial role in the atheroprotective action of 17beta-estradiol in low-density lipoprotein receptor-deficient mice[END_REF][START_REF] Billon-Galés | The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta-estradiol[END_REF] . αSMACreER T2-ERα lox/lox (control mice) and αSMACreER T2+ ERα lox/lox mice were injected daily with tamoxifen (1 mg/mouse per day, Sigma, France) during 5 days from 4 weeks of age to induce activation of the Cre recombinase. Throughout all protocols, mice were housed at the animal facility of the faculty of medicine (Toulouse, France). Mice were housed in a temperature-controlled room with a 12:12-hour light-dark cycle and maintained with access to food and water ad libidum. Details about randomization, blinding and group size are described in Online Supplementary Data.

Ovariectomy and treatments.

Bilateral ovariectomy was performed at 4 weeks of age after anesthesia with a mixture of ketamine (100 mg/kg, Merial, Lyon, France) and xylazine (10 mg/kg; Sigma-Aldrich; Isle d'Abeau Chesnes, France). After two weeks of recovery, mice were subcutaneously implanted with pellets releasing either tamoxifen (TAM, 5 mg for 60 days, i.e. 4mg/kg/d; Innovative Research of America, Sarasota, FL), 17β-Estradiol (E2, 0.1 mg for 60 days, i.e. 80 µg/kg/d; Innovative Research of America, Sarasota, FL) or vehicle (Veh). These doses of E2 and TAM were previously used effectively to demonstrate their beneficial actions on the arterial wall [START_REF] Smirnova | The Activation Function-1 of Estrogen Receptor Alpha Prevents Arterial Neointima Development Through a Direct Effect on Smooth Muscle Cells[END_REF][START_REF] Fontaine | Tamoxifen elicits atheroprotection through estrogen receptor α AF-1 but does not accelerate reendothelialization[END_REF][START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF][START_REF] Toutain | Estrogen receptor alpha expression in both endothelium and hematopoietic cells is required for the accelerative effect of estradiol on reendothelialization[END_REF][START_REF] Billon-Galés | The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta-estradiol[END_REF][START_REF] Pare | Estrogen receptor-alpha mediates the protective effects of estrogen against vascular injury[END_REF][START_REF] Karas | Effects of estrogen on the vascular injury response in estrogen receptor alpha, beta (double) knockout mice[END_REF] . We systematically checked that vehicle-treated ovariectomized mice had an atrophied uterus (<10 mg) and non-detectable circulating levels of E2, whereas those implanted with E2 or TAM releasing pellet had a significant increase of uterine weight. Mice were submitted to carotid artery injury after 3 weeks of drug administration.

Mouse carotid injury and quantification of re-endothelialization.

Endovascular injury of the carotid artery was performed on mice as described previously 27 . Briefly, the left common carotid artery was isolated and the external carotid was incised. A 0.35 mm diameter flexible wire with a 0.25 mm tip was advanced and pulled back two times into common carotid artery (5 mm length in total) and blood flow was restored. Five-days post-injury, carotid arteries were stained with Evans blue dye (sc-203736, Santa Cruz) and mounted with Kaiser's Glycerol gelatin (Merck). Electric injury of the carotid artery was performed on mice as described previously [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF]27 . Briefly, the left common carotid artery was isolated, and electric injury was applied (3 mm total length) with a bipolar microregulator. Images were acquired using DMR 300 Leica microscope using LAS V3.8 and ImageJ software. The percentage of re-endothelialization was calculated relative to the initial deendothelialized area (day 0).

Ex vivo re-endothelialization of mouse carotid artery.

Ex vivo endovascular injury of the carotid artery was performed as described previously [START_REF] Lupieri | Smooth muscle cells-derived CXCL10 prevents endothelial healing through PI3Kγdependent T cells response[END_REF] . Briefly, carotid arteries were isolated from mice and a 2mm-portion of artery was de-endothelialized with a 0.35mm diameter flexible wire with a 0.25mm tip. The carotid arteries were then flushed with endothelial growth cell medium MV (CC-22020, Promocell) medium supplemented with a supplement Mix (CC-39225, Promocell) and cultured in this medium for up to 5 days at 37°C, 5% CO2, renewing the medium every 2 days.

En face Immunostaining.

Carotid arteries were fixed for 20 min in 4% paraformaldehyde, opened longitudinally and were then permeabilized for 15 min with PBS containing 0.3% TritonX100. After blocking with PBS containing 3% BSA, 1% FBS and 0.1% tween-20 for 1hour, carotids arteries were immunostained with primary antibody (CD31 antibody (sc-1506, Santa Cruz, catalog # sc1506; 1:300), anti-VE-Cadherin antibody (555289, BD Pharmingen, catalog #555289; 1:300) for ECs and anti-αSMA (abcam-7817, catalog #7817; 1:300) for SMCs were performed overnight at 4°C. Then, the arteries were incubated with secondary antibodies for 2h (anti-mouse (Alexa Fluor 488, catalog#715-545-150, 1:300) or anti-rat (Alexa Fluor 594, #714-585-150, 1:300) for 1 hour at room temperature. Nuclei were stained with DAPI (5 µg/mL). Control experiments using the appropriate secondary antibody alone was performed to determine antibody specificity. Microscopy imaging was performed with a Zeiss LSM780 confocal microscope. Blinded imaging and quantification were performed with ImageJ software using identical exposures and matched imaging and processing conditions.

RNA sequencing and analysis.

RNA was isolated from carotid arteries through the phenol-chloroform method using TRIzol (Ambion) reagent. The resulting RNA quality was determined using a Fragment Analyzer Instrument and all samples had RNA integrity numbers above 7.4. RNA sequencing libraries were prepared according to Illumina's protocols using the Illumina TruSeq Stranded mRNA kit (ref #20020595). Eleven cycles of PCR were applied to amplify the libraries. Library quality was assessed using a Fragment Analyzer Instrument and libraries were quantified by QPCR using the kit KAPA KK4824, on the QuantStudio 6 of Thermo Fisher Scientific. Sequencing was performed in paired-end (2x150 bp) on an Illumina NovaSeq sequencer at the Integragen company platform (https://www.integragen.com/fr/). All data and materials (Fastq files) have been made publicly available at the GEO portal [START_REF] Barrett | NCBI GEO: archive for functional genomics data sets--update[END_REF] and can be accessed at GSE154268 accession number.

The reads were first trimmed for adapters and low-quality ends by the TrimGalore! Algorithm (--t, -q, -e, --length 20) available on the Galaxy web server (https://usegalaxy.org/). Subsequent informatics processes of the sequenced reads were locally done under python and R environments. Reads were aligned onto the mouse genome (mm10) by bowtie2 (--fr, --no-mixed, --no-discordant, --sensitive) [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] . Reads were further processed by the featureCounts tool (-B, -C, -O) (Lio et al., 2013) to evaluate gene expression levels using the mus_musculus. GRCm38.98 Ensembl version of genes' annotations. All sequencing statistics are provided within the (Online Table I).

Primary evaluation of the 18 samples expression profiles by PCA (plot within Online Figure 1) evidenced that one OVX and one TAM libraries were far from being grouped with their corresponding ones. The functional annotations of the genes exhibiting a 2-fold different expression level in these samples as compared to their relative clearly indicated that these 2 samples were contaminated by mRNAs of neural origin (Online Table II), presumably from the vagus nerve located near the carotid. Discarding these 2 samples allowed the rest of the samples to be correctly grouped by the PCA evaluation (Online Figure I). Differential gene expression analysis was next performed using the R-interfaced n o t 6 DESeq2 suite [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF] following the removal of genes with no detected expression. We used the HTSfilter [START_REF] Rau | Data-based filtering for replicated high-throughput transcriptome sequencing experiments[END_REF] to set the threshold values to filter the weakly expressed genes. Genes were declared as differentially regulated when their FC was > 1.5 or < 0.66 with a BH adjusted P-value < 0.05. Functional annotations were made using the GSEA program (v4.0.3) 33 interrogating MSigDB hallmarks [START_REF] Liberzon | The Molecular Signatures Database (MSigDB) hallmark gene set collection[END_REF] and the EnrichR web platform (https://amp.pharm.mssm.edu/Enrichr/) [START_REF] Kuleshov | Enrichr: a comprehensive gene set enrichment analysis web server 2016 update[END_REF] .

Gene expression analysis.

Tissues (carotid artery, aorta and enriched SMCs fraction) were homogenized using a Precellys tissue homogenizer (Bertin Technol., Cedex, France) and total RNA from tissues was prepared using TRIzol (Invitrogen, Carlsbad, CA). 500 ng of RNA was reverse transcribed (RT) at 25°C for 10 min and then at 37°C for 2 h in 20 µl final volume using the High Capacity cDNA reverse transcriptase kit (Applied Biosystems). For gene expression, qPCR was performed using SsoFast EvaGreen Supermix (Bio-Rad) on the StepOne instrument (Applied Biosystems). Primers validated by testing the PCR efficiency using standard curves (95% < efficiency < 105%). Gene expression was quantified using the comparative C t (threshold cycle) method; tumor protein, translationally-controlled 1 (Tpt1) was used as reference. Details about Q-PCR conditions are described in Online Supplementary Data.

Statistical analysis.

Data are shown as means ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA; www.graph pad.com). A difference of P<0.05 was considered significant. Gaussian (normal) distribution was determined using Shapiro-Wilks normality test (n≥5). For normally distributed populations, student's t-test (2 groups) or 1-way ANOVA followed by Tukey's post-test (3 or more groups) was conducted. To test the respective roles of treatment and genotype, a 2-way ANOVA was performed. In case of significant interaction, Tukey's post-test was subquently performed. For data that failed normality testing (including n≤5), Mann-Whitney test (2 groups) or Kruskal-Wallis with Dunn's post-test (3 or more groups) was performed. No corrections for multiple testing were made across assays. Exact number (n), precise P values and statistic test used in each experiment are described in Online Supplemental Data. When "representative images" are shown, the selected images were those that most accurately represented the average data obtained in all the samples.

RESULTS

Tamoxifen accelerates endothelial healing and this action requires the presence of underlying smooth muscle cells.

To evaluate the effect of TAM treatment on endothelial healing, we first used a mouse model of endovascular injury of the carotid in vivo 27 . To this aim, ovariectomized wild-type female mice were chronically treated with either vehicle or TAM (Figure 1A). E2 was used as a positive control of full ERα activation. As expected, both chronic treatment with E2 or TAM led to uterine hypertrophy, confirming the agonist effect of TAM alone in this organ (Figure 1B). Indeed, TAM and E2 both led to an increase in uterine weight gain (100 mg ± 7.93 and 50 mg ± 2.46 respectively). Endothelial healing rates were analyzed and quantified after Evans Blue (Figure 1C) or CD31 (Figure 1D) en face staining 5 days after arterial endovascular injury in these mice. Quantification of re-endothelialized area showed 30 % of endothelial regeneration in vehicle-treated mice compared to about 70 % in both E2 and TAM treated female mice (Figures 1C and1D). Interestingly, tamoxifen also accelerated endothelial healing in intact or castrated male mice with the same efficacy than in female mice (Online Figure II). In ovariectomized female mice, combination of E2 and TAM treatment induced a similar acceleration of endothelial healing than E2 or TAM alone (Figure 1E). This contrasts with the partial antagonist action of TAM on uterus. Indeed, as previously described [START_REF] Johnston | Idoxifene antagonizes estradiol-dependent MCF-7 breast cancer xenograft growth through sustained induction of apoptosis[END_REF] , animals treated with a combination of E2 plus TAM had significantly smaller uterine weights than the ones treated with E2 alone (64.2 mg ± 4.1 versus 100 mg ± 7.93) (Online Figure III). To evaluate the regenerated endothelium after complete re-endothelialization, we performed VE-cadherin staining of the arteries 10 days after injury. The VE-cadherin staining pattern of reendothelialized area was similar in vehicle and treated mice, suggesting the efficiency of functional integrity of the endothelium after healing, regardless of the treatment (Figures 1F and1G).

These findings are in contrast with our previous work reporting that TAM failed to accelerate endothelial healing after perivascular electric injury [START_REF] Fontaine | Tamoxifen elicits atheroprotection through estrogen receptor α AF-1 but does not accelerate reendothelialization[END_REF] . We first confirmed that TAM, unlike E2, does not accelerate endothelial healing after electric injury of the carotid artery (Figures 2A and2B). We previously demonstrated that E2 effect on endothelial healing after electric injury is mediated by membrane ERα [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF] . The fact that TAM was not able to elicit this membrane ERα action raises two hypotheses: 1) either TAM fails to bind membrane ERα, or 2) TAM is able to bind but does not activate membrane ERα. To address this question, we co-administrated E2 and TAM and found no acceleration of endothelial healing after electric injury of the carotid from these mice compared to vehicle. Thus, TAM has an antagonist activity on membrane ERα signaling pathway.

In addition, endovascular and perivascular injuries of the mouse carotid artery are similar in their efficiency to destroy the endothelium, but they differ since endovascular, but not perivascular injury preserves the medial smooth muscle cells [START_REF] Brouchet | Estradiol accelerates reendothelialization in mouse carotid artery through estrogen receptor-alpha but not estrogen receptor-beta[END_REF]27,[START_REF] Carmeliet | Vascular wound healing and neointima formation induced by perivascular electric injury in mice[END_REF][START_REF] Lindner | Mouse model of arterial injury[END_REF] (Online Figures IV andV). Our result thus demonstrates, that TAM accelerates endothelial healing but, at variance to E2, its action requires smooth muscle cells integrity. Recently, two studies highlighted the importance of SMCs-ECs paracrine communication during this regenerative process [START_REF] Lupieri | Smooth muscle cells-derived CXCL10 prevents endothelial healing through PI3Kγdependent T cells response[END_REF][START_REF] Jun | Novel Paracrine Functions of Smooth Muscle Cells in Supporting Endothelial Regeneration Following Arterial Injury[END_REF] . Depending on the context, SMCs could either promote re-endothelialization through CXCL7-mediated recruitment of ECs [START_REF] Jun | Novel Paracrine Functions of Smooth Muscle Cells in Supporting Endothelial Regeneration Following Arterial Injury[END_REF] or on the contrary prevent this process via CXCL10 secretion [START_REF] Lupieri | Smooth muscle cells-derived CXCL10 prevents endothelial healing through PI3Kγdependent T cells response[END_REF] . However, no difference in either Cxcl7 or Cxcl10 mRNA levels were observed in TAM or E2 treated mice (Online Figure VI).

TAM, in contrast to E2, is still able to accelerate endothelial healing after endovascular injury ex vivo.

Endothelial healing also involves the coordinated action of circulating immune cells [START_REF] Lupieri | Smooth muscle cells-derived CXCL10 prevents endothelial healing through PI3Kγdependent T cells response[END_REF][START_REF] Newby | An overview of the vascular response to injury: a tribute to the late Russell Ross[END_REF] . To investigate the possible contribution of circulating factors in TAM action during re-endothelilization process, we used a model of ex vivo endothelial healing [START_REF] Lupieri | Smooth muscle cells-derived CXCL10 prevents endothelial healing through PI3Kγdependent T cells response[END_REF] . De-endothelialization was performed ex vivo and endothelial healing was evaluated after 5 days of culture in an EC-specific medium. α-SMA immunostaining and contractile response to KCl revealed that SMCs are viable and functional at the end of the procedure (Online Figure VII). Experiments performed on carotid arteries from mice treated with either vehicle, E2 or TAM indicated that in vivo "preconditioning" with TAM, but not with E2, was sufficient to accelerate endothelial healing ex vivo (Figure 2D). This in vivo pre-treatment of the mice with TAM was necessary since ex vivo treatment of the carotid artery with 4-hydroxytamoxifen (4-OHT), the main active metabolite of TAM, was unable to accelerate endothelial healing. Altogether, these results concur to demonstrate that i) the cellular actors involved in E2 and TAM endothelial healing are quite different and ii) a period of in vivo TAM exposure before the injury is necessary and sufficient to subsequently accelerate endothelial healing (Online Figure VIII), suggesting long-term genomic actions as explored in the next paragraph.

Chronic E2 and TAM treatment induces different gene expression profiles in the carotid artery.

We therefore reasoned that performing RNA sequencing on non-injured carotid from ovariectomized mice treated chronically with E2 or TAM may help to identify a subset of genes involved in this effect. Gene regulations by E2, TAM and vehicle in the carotid artery were compared and presented in the heatmap (Figure 3A). The number of significantly regulated genes at a fold-change >1.5 over vehicle level was higher after E2 treatment than after TAM exposure, especially for up-regulated n o t 8 genes. Importantly, the patterns of genes regulated by E2 and TAM (absolute fold change >1.5; P value <0.05) were substantially different (Figure 3B). Indeed, only 68 genes were commonly regulated by these two ER ligands from totals of 717 and 227 genes regulated by E2 and TAM, respectively. Independent RT-qPCR quantification confirmed the changes indicated by RNAseq analysis, except for some genes (Tgm3, Tent5b, Aqp5) which were found to be regulated by TAM but not by E2 (Online Figure IX). Of note for these genes, RNAseq analysis indicated a more pronounced effect of TAM (log2fold changes: 3.2, 1.6 and 4.5) than of E2 (log2fold changes: 0.7, 0.7 and 1.8). Moreover, the use of ERα -/-mice highlighted that most of the regulated genes were ERα dependent since transcriptional regulation in response to TAM was totally abolished (Online Figure X).

Functional annotation of the gene subsets regulated by both E2 and TAM by the GSEA method 33 revealed MSigDB 34 hallmarks for early and late estrogen response including classical estrogenresponsive genes such as Pgr. In addition, as revealed by previous large scale approaches performed in 41,42 the aorta in response to E2, we found that Grem2 was up-regulated by E2 and also by TAM. Interestingly, Hedgehog (Hh) signalling was the only hallmark specifically enriched for TAM-regulated genes in non-injured carotid arteries (Figure 3C). Furthermore, expression of Gli1/2 and Ptch1/2, the main readouts of Hh signalling activity was up-regulated in the carotid arteries of TAM-treated mice compared to vehicle-treated mice during the re-endothelialization process i.e 1, 3 or 5 days after endovascular injury (Online Figure XI A). Since Hh pathway is well described to be involved in angiogenesis, in particular through Vegf upregulation [START_REF] Renault | Desert hedgehog promotes ischemia-induced angiogenesis by ensuring peripheral nerve survival[END_REF][START_REF] Chen | Administration of sonic hedgehog protein induces angiogenesis and has therapeutic effects after stroke in rats[END_REF][START_REF] Pola | The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors[END_REF] , we analyzed genes linked this pathway during endothelial healing process. Interestingly, we found that Vegfa, Vegfd and Flk1 mRNA levels were higher in the carotid from TAM-treated mice compared to those from the control group (vehicle-treated mice) (Online Figure XI B). We then took advantage of the published transcriptome single-cell analysis obtained on mouse aorta [START_REF] Kalluri | Single Cell Analysis of the Normal Mouse Aorta Reveals Functionally Distinct Endothelial Cell Populations[END_REF] , and comparison of these data with ours indicated that a majority of the genes that we identified as regulated by E2 in the carotid arteries were mainly expressed in monocytes and fibroblasts, while those responding to TAM are representative of genes expressed in SMCs and fibroblasts (Online Figure XII).

Altogether, these results revealed that gene expression profiles in carotid arteries from mice treated by TAM and E2 substantially differ both in terms of regulated pathways and of cellular targets.

In contrast to E2, TAM effect on endothelial healing requires smooth muscle cells (SMCs) ERα but not endothelial/hematopoietic ERα

Thus, we used the Cre-lox strategy (Tie2Cre ERα lox/lox and αSMACreER T2 ERα lox/lox mice) to directly evaluate the role of ERα in endothelial/hematopoietic cells versus smooth muscle cells respectively. The efficiency of treatment with either E2 or TAM was verified by the uterine response in each mouse model (Online Figure XIII). First, we showed that E2 regenerative effect on endothelium after endovascular injury was abolished in Tie2Cre+ERα lox/lox mice, demonstrating the crucial role of ERα in hematopoietic/endothelial compartment in response to E2 in this model of endovascular injury, similar to that observed for perivascular electric injury [START_REF] Toutain | Estrogen receptor alpha expression in both endothelium and hematopoietic cells is required for the accelerative effect of estradiol on reendothelialization[END_REF] . In contrast, TAM effect on endothelial healing was fully preserved in Tie2Cre+ERα lox/lox mice (Figure 4A). Then, we used a mouse model expressing the inducible Cre-ER T2 fusion gene under the control of the αSMA promoter with ERα lox/lox mice to evaluate whether SMCs ERα is involved in endothelial healing after mechanical injury in response to TAM. In both genotypes, vehicle-treated mice displayed a similar low percentage of re-endothelialization, close to 40 % of re-endothelialization. As expected, both E2 and TAM treatment increased endothelial healing in control mice, raising 80 % of re-endothelialization. In αSMACreER T2 + ERα lox/lox mice, TAM failed to have such an effect, by contrast to the fully preserved effect of E2 (50 % of re-endothelialization in TAM and vehicle-treated mice as compared to 80 % in E2-treated mice) (Figure 4B). Altogether, these results identified SMCs as the main target cells for the accelerative effect of TAM on endothelial healing.

d i s t r i b u t e .

9 Importantly, this result highlights that, although the accelerative action on endothelial healing by E2 and TAM are both mediated through ERα, the cellular targets involved in this process are completely different.

Whereas E2 action on endothelial healing requires ERαMISS, this effect is mediated by nuclear ERα in response to TAM.

We previously showed that membrane ERα mediates acceleration of re-endothelialization after electric injury in response to E2 [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF] independently of nuclear ERα [START_REF] Billon-Galés | Activation function 2 (AF2) of estrogen receptoralpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing[END_REF] . Using a mice model harboring a mutation of ERα cysteine 451, recognized as the key palmitoylation site required for ERα plasma membrane location (ERα-C451A), we confirmed the crucial role of membrane ERα in response to E2 in this process using the model of endovascular injury. Indeed, we observed no effect of E2 on endothelial healing in ERα-C451A mice. By contrast, the effect of TAM was fully preserved in this mouse model of loss of membrane localization of ERα (Figure 5A Because ERαAF1 is necessary to mediate actions of TAM in vitro [START_REF] Berry | Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4hydroxytamoxifen[END_REF]49 and in vivo [START_REF] Abot | The AF-1 activation function of estrogen receptor α is necessary and sufficient for uterine epithelial cell proliferation in vivo[END_REF][START_REF] Guillaume | Selective Activation of Estrogen Receptor α Activation Function-1 Is Sufficient to Prevent Obesity, Steatosis, and Insulin Resistance in Mouse[END_REF] , we then sought to evaluate the role of this sub-function on endothelial healing. To this aim, ovariectomized ERαAF1 +/+ and ERαAF1 0 mice were treated with E2, TAM or a vehicle, and were submitted to mechanical injury of the carotid. E2 treatment increased endothelial healing in both ERαAF1 +/+ and ERαAF1 0 control mice, extending the previous results obtained in the perivascular model [START_REF] Billon-Galés | The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta-estradiol[END_REF] . TAM failed to have such an effect using ERαAF1 0 mice, highlighting the crucial role of ERαAF1 in the vascular response to TAM (Figure 5B). Accordingly, RT-qPCR analysis performed in the aorta of these mice revealed the crucial role of ERαAF1 in the transcriptional action of TAM since the induction of the most regulated ERα target genes in response to TAM was abrogated in ERαAF1 0 mice (Figure 6A). Importantly, analysis of this gene regulation profile in the smooth muscle cell enriched-fraction (media) further reinforces the importance of SMCs in the transcriptional response of TAM in the vessel (Figure 6B). Altogether, our data indicate that, E2 and TAM act via the same receptor: ERα healing but these two ligands target different sub-functions of the receptor: membrane ERα for E2 and nuclear ERαAF1 in response to TAM.

DISCUSSION

Clinical studies report a protective action of TAM on cardiovascular diseases but mechanisms underlying the beneficial agonist action of this drug remain poorly understood. The main aims of this study were to evaluate the action of TAM on endothelial healing and to explore the cellular and molecular targets involved. Here, we demonstrated for the first time that TAM accelerated endothelial healing after endovascular injury in both female and male mice. By contrast, in the model of perivascular electric injury, in which medial smooth muscle cells are destroyed, TAM failed to accelerate endothelial healing, suggesting an important role of this cell type in TAM action. Accordingly, we found that ERα in SMCs is necessary to mediate this vascular action. Medial SMCs could be critical in orchestrating vascular injury response through paracrine action. Indeed, SMCs continuously dialogue with ECs. However, whether and how SMCs may influence endothelial repair remains largely unknown. Recently, it was reported that SMCs promote re-endothelialization through CXCL7-mediated recruitment of ECs from uninjured endothelium [START_REF] Jun | Novel Paracrine Functions of Smooth Muscle Cells in Supporting Endothelial Regeneration Following Arterial Injury[END_REF] . Lupieri et al. also demonstrated that SMCs through CXCL10 prevent endothelial healing 28 . Thus, depending on the context, SMCs paracrine factors can accelerate or slow endothelial healing. However, no change in the profile of expression of these cytokines could be detected under E2 or TAM treatment in both intact and injured carotids.

The major findings of this study are that TAM can mimic estrogen vasculoprotective effects through the activation of different/divergent cellular and molecular targets. Indeed, both E2 and TAM can accelerate endothelial healing but this beneficial action is dependent on the experimental model (i.e. dependent on the cell type environment of the model used). Indeed, TAM but not E2 required the presence of the underlying medial SMCs (comparison of perivascular versus endovascular injuries). In addition, the vivo pre-treatment with TAM, but not with E2, is sufficient to induce acceleration of endothelial healing after ex vivo injury. Accordingly, Cre-Lox models reinforce this conclusion since we demonstrated that TAM action is mediated by ERα in SMCs, whereas E2 action relies on endothelial/hematopoietic ERα. Hence, transcriptomic analysis reveals differential gene expression profiles in the carotid artery from mice treated chronically with E2 or TAM. Functional annotation of the gene subsets regulated by either ER ligand indicated that inflammatory response was the major MSigDB 34 hallmark enriched for E2 regulated genes but not in response to TAM. The Hedgehog signaling pathway was specifically enriched for TAM-regulated genes in intact carotid arteries. The expression of several genes involved in the Hedgehog signaling pathway were also significantly higher in carotid from TAM-treated mice compared to control mice during the endothelial healing process (1, 3 or 5-day postinjury). This is in accordance with previous works in adult blood vessels, showing in several experimental models that activation of this pathway promotes endothelial wound healing and angiogenesis through an indirect action on endothelial cells exerted by SMCs and/or fibroblasts [START_REF] Renault | Desert hedgehog promotes ischemia-induced angiogenesis by ensuring peripheral nerve survival[END_REF][START_REF] Chen | Administration of sonic hedgehog protein induces angiogenesis and has therapeutic effects after stroke in rats[END_REF][START_REF] Pola | The morphogen Sonic hedgehog is an indirect angiogenic agent upregulating two families of angiogenic growth factors[END_REF][START_REF] Asai | Topical sonic hedgehog gene therapy accelerates wound healing in diabetes by enhancing endothelial progenitor cell-mediated microvascular remodeling[END_REF][START_REF] Agouni | Sonic hedgehog carried by microparticles corrects endothelial injury through nitric oxide release[END_REF] . Interestingly, the comparison of genes selectively regulated by TAM with the single-cell data on mouse aorta [START_REF] Kalluri | Single Cell Analysis of the Normal Mouse Aorta Reveals Functionally Distinct Endothelial Cell Populations[END_REF] revealed that these genes are mainly expressed in SMCs. By contrast, genes regulated by E2 are reminiscent of the expression pattern of immune cells.

SERMs, such as TAM, are defined as molecules mimicking the actions of E2 in some tissues and antagonizing the actions of E2 in others. The main mechanism underlying this tissue-specificity was so far attributed to differential cofactor expression/recruitment in each tissue. In the breast, where TAM is an antagonist, TAM induces the recruitment of co-repressors instead of co-activators to ER target promoters 11 . In contrast, in the uterus where TAM acts as an agonist, it recruits co-activators instead of corepressors to ER target genes [START_REF] Shang | Molecular determinants for the tissue specificity of SERMs[END_REF] . The present results highlight, for the first time, to our knowledge, that the cell/tissue specificity of SERM may also involve different cell types of ERα, shedding a new light on the mechanisms of action of Selective Modulation of ERα. This new dimension will therefore have to be taken into account when examining the mechanisms underlying the benefit/risk profile of TAM for the treatment of breast cancer. Among other interrogations raising from our finding, since ERαregulates the immune system, it appears crucial to (re)-consider the action of TAM on immune surveillance in cancer 54 . Interestingly, estrogens and TAM, but not pure antiestrogens, were also recently shown to partially protect against severe acute respiratory syndrome (SARS) in a mouse-adapted SARS-CoV model [START_REF] Channappanavar | Sex-Based Differences in Susceptibility to Severe Acute Respiratory Syndrome Coronavirus Infection[END_REF] .

We have developed for two decades several mouse models of carotid artery injury. Thanks to the combination of i) genetically models targeting ERα or its sub-functions and ii) pharmacological tools, we were able to demonstrate that acceleration of endothelial healing in response to E2 is entirely dependent on membrane ERα [START_REF] Adlanmerini | Mutation of the palmitoylation site of estrogen receptor α in vivo reveals tissue-specific roles for membrane versus nuclear actions[END_REF][START_REF] Billon-Galés | Activation function 2 (AF2) of estrogen receptoralpha is required for the atheroprotective action of estradiol but not to accelerate endothelial healing[END_REF][START_REF] Abot | The uterine and vascular actions of estetrol delineate a distinctive profile of estrogen receptor α modulation, uncoupling nuclear and membrane activation[END_REF] . In line with our previous work [START_REF] Fontaine | Tamoxifen elicits atheroprotection through estrogen receptor α AF-1 but does not accelerate reendothelialization[END_REF] , we first confirmed that TAM failed to activate membrane ERα, raising the question of whether this is due to the failure of TAM to bind to membrane ERα or the failure of membrane ERα to become activated by TAM binding. Co-administration of TAM and E2 validated the later hypothesis, as it failed to accelerate endothelial healing after electric injury, showing that TAM is not only devoid of ERαMISS, but TAM is also able to antagonize these E2 MISS effects. The role of this membrane/extra-nuclear action of TAM in another pathophysiological context, in particular, breast cancer angiogenesis 57 should be studied in future works. The inhibition of the migration and invasion of breast cancer cells induced by extra-nuclear signaling in response to estrogen would be of peculiar interest [START_REF] Giretti | Extra-nuclear signalling of estrogen receptor to breast cancer cytoskeletal remodelling, migration and invasion[END_REF] .

The use of mice harboring mutated specific functions of ERα revealed that TAM acts through nuclear ERα activation, and more precisely through the AF1 to accelerate endothelial healing. It is highly likely that the nuclear ERαAF1 activation by tamoxifen occurred prominently in SMCs, but in the absence of cell specific invalidation of this function does not allow to affirm that. In vitro experiments previously demonstrated the crucial role of AF1 to the agonist action of TAM [START_REF] Berry | Role of the two activating domains of the oestrogen receptor in the cell-type and promoter-context dependent agonistic activity of the anti-oestrogen 4hydroxytamoxifen[END_REF]49 . More recently, we d i s t 11 showed that ERαAF1 is also necessary in vivo to relay the effects of TAM on: i) the proliferation of uterine epithelial cells [START_REF] Abot | The AF-1 activation function of estrogen receptor α is necessary and sufficient for uterine epithelial cell proliferation in vivo[END_REF] , ii) the prevention of atherosclerosis 9 , and iii) the prevention of high-fat dietinduced metabolic disorders [START_REF] Guillaume | Selective Activation of Estrogen Receptor α Activation Function-1 Is Sufficient to Prevent Obesity, Steatosis, and Insulin Resistance in Mouse[END_REF] , since all these beneficial actions are abrogated using mouse model selectively deficient for ERαAF1 (ERαAF1 0 ). Interestingly, ERαAF1 is dispensable to mediate these two latter actions in response to E2 [START_REF] Billon-Galés | The transactivating function 1 of estrogen receptor alpha is dispensable for the vasculoprotective actions of 17beta-estradiol[END_REF][START_REF] Handgraaf | Prevention of obesity and insulin resistance by estrogens requires ERα activation function-2 (ERαAF-2), whereas ERαAF-1 is dispensable[END_REF] .

To conclude, the present study demonstrates that TAM and E2 both accelerate endothelial healing through an ERα-dependent effect. However, the cellular (ECs versus SMCs) and molecular (ERα subfunctions) target accounting for this protective action are quite dissimilar. These results should serve as a paradigm to revisit the action of estrogens and SERMs in breast cancer initiation and growth, angiogenesis and immune surveillance, but also in arterial pathophysiology. Although successfully used for 40 years in medicine, the understanding of the mechanisms of action of TAM are still in their beginning. Its arterial protective actions should be seriously considered in the evaluation of benefit/risk ratio in the treatment of breast cancer.

F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

13 mouse models. Four weeks old mice were ovariectomized and received E2 or TAM treatment for 3 weeks. Mice were then submitted to endovascular injury of the carotid artery (day 0). De-endothelialized area was evaluated after blue Evans staining. Quantitative analysis of reendothelialization was expressed as a percentage of re-endothelialized area at day 5 compared to day 0. The outlined zones correspond to the de-endothelialized area. Scale bar, 500µm. Results are expressed as means ± SEM (n=5-14 per group). To test the effect of E2 or TAM treatments in each genotype, Kruskall-Wallis test were performed (*P<0.05, **P<0.01, ***P<0.001).

F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e . F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

F o r 18

NOVELTY AND SIGNIFICANCE

What Is Known?

 The main estrogen, 17β-estradiol (E2) accelerates endothelial healing by targeting membrane ERα in endothelial cells.  Tamoxifen is a selective ER modulator that acts as an agonist or antagonist depending on the tissue.  Tamoxifen is used as an adjuvant therapy against breast cancer and its effect on the vascular system remains to be better understood.

What New Information Does This Article Contribute?

 As E2, Tamoxifen is able to accelerate endothelial healing, but in contrast to E2:  Tamoxifen accelerates endothelial healing indirectly by targeting ERα in smooth muscle cells.  Tamoxifen accelerates endothelial healing by targeting nuclear ERα, and more specifically the activation function 1.

Cardiovascular diseases have become an increasingly important cause of mortality among breast cancer survivors. Millions of breast cancer patients received Tamoxifen as adjuvant treatment for at least 5 years but its effect on the vascular system remains to be better defined. Indeed, although successfully used for 40 years in medicine, the understanding of its mechanisms of action in vivo is only now beginning to emerge. Tamoxifen acts as a selective modulator of the estrogen receptor (SERM), antagonizing or mimicking 17β-estradiol (E2) action depending on the tissues. We show here that as E2, Tamoxifen contributes to vascular protection through acceleration of endothelial healing. Surprisingly, even though both E2 and Tamoxifen actions on endothelial healing are mediated by ER, activation of this receptor occurs in different cell types. In contrast to E2, which activates membrane ERα in endothelial cells, Tamoxifen accelerates endothelial healing by targeting nuclear ERα in smooth muscle cells. These results should serve as a paradigm to revisit the action of SERMs not only in arterial pathophysiology but also in angiogenesis and breast cancer. The spectrum of the vascular protective actions of Tamoxifen should also be reconsidered in the assessment of benefit/risk ratio in the treatment of breast cancer. A.

F o r C i r c u l a t i o n R e s e a r c h P e e r R e v i e w . D o n o t d i s t r i b u t e . D e s t r o y a f t e r u s e .

Injured carotid Non injured carotid

B.

ERa-WT ERα-C451A

ERα-AF1 0

Figure 1 .

 1 Figure 1. As E2, TAM induces acceleration of endothelial healing after endovascular injury in vivo.(A) Four-week-old female mice were ovariectomized and 2 weeks after received either vehicle, E2 (80 µg/kg/day), TAM (4 mg/kg/day) or combination of both E2 and TAM. Three weeks later, mice were submitted to endovascular injury of the carotid artery. Carotid re-endothelialization was analyzed 5 days after injury. (B) Representative photography of uterus. (C) Representative Evans blue or (D) CD31+ staining of carotids (Scale Bar, 500µm) and (E) quantitative analysis of re-endothelialization, expressed as a percentage of re-endothelialized area compared to day 0. The outlined zones correspond to the deendothelialized area. Results are expressed as means ± SEM (n=5-12 per group). Carotids were also analyzed 10 days after the injury: (F) Immunostaining of endothelial cells with anti-VE-cadherin antibody day 10 after endovascular injury (Scale Bar, 20µm) and (G) quantitative analysis for VE-cadherin fluorescent intensity. Results are expressed as means ± SEM (n=4-6 per group). To test the effect of the different treatments, Kruskall-Wallis test were performed (**P<0.01, ***P<0.001).

Figure 2 .

 2 Figure 2. The cellular actors involved in the acceleration of endothelial healing in response to TAM and E2 are different.Four-week-old female mice were ovariectomized and 2 weeks after were given either vehicle, E2 (80 µg/kg/day), TAM (4 mg/kg/day) or combination of both E2 and TAM. (A) Three weeks later, mice were submitted to electric (perivascular) injury of the carotid artery. (B) Representative image of Evans bluestained en face carotid arteries from indicated treatment (left panel) and quantitative analysis of reendothelialization expressed as a percentage of re-endothelialized area compared to day 0 (right panel). Scale bar, 500µm. (C) Three weeks later, carotid arteries were isolated and de-endothelialized ex vivo. Subsequently, they were placed in an endothelial cell-specific medium for 5 days. (D) CD31 staining of en face carotid arteries. The zone outlined in yellow represents the area that has not been recovered with endothelial cells. Scale bar, 450µm. Quantification of re-endothelialization expressed as the percentage of CD31+ area compared to day 0. Results are expressed as means ± SEM (n=6-12 per group). To test the effect of the different treatments, Kruskall-Wallis test (abnormal distribution) (B) or one-way ANOVA (normal distribution) (D) were performed (*P<0.05). † indicates differences between E2 and TAM or E2+TAM ( † P<0.5, † † P<0.01).

Figure 3 .

 3 Figure 3. E2 and TAM regulate a different subset of genes in carotid arteries.(A) RNAs were isolated from carotid arteries of ovariectomized mice (n=6 per group) treated by E2 (80 µg/kg/day) or TAM (4 mg/kg/day) for 3 weeks and sequenced. The heatmap shown illustrates the relative expression values of all genes significantly regulated following E2 and/or TAM treatment (threshold of fold-change of expression >1.5 over control with a BH corrected p-value < 0.05). HCL clustering regroups each sample with its corresponding treatment group. (B) Venn diagram representing the overlap of genes regulated by E2 and/or TAM in carotid arteries. (C) GSEA analysis representing the different hallmarks pathways regulated by E2 and TAM. Calculated FDR q-value is given for each term, as well as the heatmap of the normalized expression values (Norm.Expr.data) of associated genes in each sample. Notably, the hallmarks specifically enriched in the E2 condition involve mainly genes regulated only by E2.

Figure 4 .

 4 Figure 4. Estrogen receptor α (ERα) in smooth muscle cells is necessary to induce endothelial healing in response to TAM but not to E2. Schematic representation of (A) Tie2CreERα lox/lox and (B) αSMACre ERT2 ERα lox/lox

Figure 5 .

 5 Figure 5. Nuclear but not membrane ERα is necessary to induce endothelial healing in response to TAM. Schematic representation of (A) ERαC451A and (B) ERαAF1 0 mouse models. Four weeks old mice were ovariectomized and received E2 or TAM treatment for 3 weeks. Mice were submitted to endovascular injury of the carotid artery (day 0). De-endothelialized area was evaluated after blue Evans staining. Quantitative analysis of re-endothelialization was expressed as a percentage of re-endothelialized area at day 5 compared to day 0. Results are expressed as means ± SEM (n=4-8 per group). To test the effect of E2 or TAM treatments in each genotype, a 2-way ANOVA (A) or a Kruskall-Wallis test (B) were performed (*P<0.05, **P<0.01, ****P<0.0001).

Figure 6 .

 6 Figure 6. ERαAF1 is necessary for the transcriptional response to TAM in the aorta and in the enriched SMCs fraction (media). mRNA levels relative to Tpt1 mRNA level from (A) aorta (B) and smooth muscle cell (SMC) enrichedfraction from ERαAF1 +/+ and ERαAF1 0 mice treated by E2 (80 µg/kg/day) or TAM (4 mg/kg/day) during 3 weeks were quantified by RT-qPCR. Results are expressed as means +/-SEM (n=4-9/group). To test the effect of TAM treatment in each genotype, Kruskall-Wallis test were performed (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

  Figure1

  Figure 3

  Figure6

ACKNOWLEDGMENTS

The authors thank the ANEXPLO-GENOTOUL platform (INSERM UMR1048, Toulouse, France) for skillful technical assistance in animal functional experimentation, the GeT-TQ Genopole platform (INSERM UMR1048, Toulouse, France) for technical contribution to RNAseq and RT-qPCR experiments and the TRI-Genotoul (light microscopy) Platform of Toulouse for en face microscopy. We thank Jordan Rivron for providing technical support regarding the measurement of SMCs contraction in ex vivo endovascular injury of the carotid and Thierry Anthouard (Arkesys) for its help on statistical analysis.

SOURCES OF FUNDING

The work at INSERM U1048 was supported by INSERM, CHU and Université de Toulouse III, Faculté de Médecine de Toulouse, ANR (EstroShear, ANR-18-CE14-0016-01), Fondation de France, Institut Universitaire de France and Region Occitanie (REGENEV). Work at the CNRS UMR6290 was supported by the CNRS and University of Rennes1.


Amina Khettabi

Arkadiusz Grempka

Frederic Lafolet

Elarbi Chatir

Nicolas Leconte

Marie-Noelle Collomb

Damien Jouvenot

Saioa Cobo
email: saioa.cobo@univ-grenoble-alpes.fr

Catalytic light-triggered reduction promoted by a dithienylethene derivative

Keywords: Photo-reduction, dithienylethene, photochromism, catalytic reduction

come

Efficient photochromic molecular materials able to trigger chemical or physical properties changes upon irradiation have attracted a lot of interest over the last years. [START_REF] Bléger | Visible-Light-Activated Molecular Switches[END_REF][START_REF] Leydecker | Flexible Non-Volatile Optical Memory Thin-Film Transistor Device with over 256 Distinct Levels Based on an Organic Bicomponent Blend[END_REF][START_REF] Irie | Review Photochromism of Diarylethene Molecules and Crystals[END_REF][START_REF] Irie | Photochromism of Diarylethene Molecules and Crystals : Memories , Switches and Actuators[END_REF][START_REF] Kobatake | Rapid and Reversible Shape Changes of Molecular Crystals on Photoirradiation[END_REF][START_REF] Roldan | Charge Transport in Photoswitchable Dimethyldihydropyrene-Type Single-Molecule Junctions[END_REF] The good resistance to fatigue and fast isomerization rates of dithienylethene (DTE) derivatives make them some of the most promising candidates for practical applications. DTE is a positive photochrom that undergoes a photocyclisation under UV light irradiation affording a molecule with increased π-conjugation. [START_REF] Irie | Review Photochromism of Diarylethene Molecules and Crystals[END_REF][START_REF] Tian | Recent Progresses on Diarylethene Based Photochromic Switches[END_REF][START_REF] Irie | Photochromism of Diarylethene Molecules and Crystals : Memories , Switches and Actuators[END_REF][START_REF] Terao | Light-Driven Molecular-Crystal Actuators: Rapid and Reversible Bending of Rodlike Mixed Crystals of Diarylethene Derivatives[END_REF] The reverse reaction leading to the DTE ring-opening is achieved by irradiation in the visible spectrum. The vast majority of studies carried out with DTE molecules have focused on the properties of the photo-isomerization process with a particular interest in the field of molecular electronics. [START_REF] Tsujioka | Nonvolatile Organic Memory Based on Isomerization of Diarylethene Molecules by Electrical Carrier Injection[END_REF][START_REF] Uchida | Reversible On / Off Conductance Switching of Single Diarylethene[END_REF][START_REF] Katsonis | Reversible Conductance Switching of Single Diarylethenes on a Gold Surface[END_REF][START_REF] Hayakawa | Optically and Electrically Driven Organic Thin Film Transistors with Diarylethene Photochromic Channel Layers[END_REF][START_REF] Kim | Charge Transport Characteristics of Diarylethene Photoswitching Single-Molecule Junctions[END_REF][START_REF] Tsujioka | Photocurrent Detection from Photochromic Diarylethene Film[END_REF][START_REF] Hayakawa | Photoisomerization-Induced Manipulation of Single-Electron Tunneling for Novel Si-Based Optical Memory[END_REF] In contrast, the redox properties of DTE derivatives have been much less explored. [START_REF] Gilat | Light-Triggered Molecular Devices: Photochemical Switching Of Optical and Electrochemical Properties in Molecular Wire Type Diarylethene Species[END_REF][START_REF] Gorodetsky | Reductive Electrochemical Cyclization of a Photochromic 1,2-Dithienylcyclopentene Dication[END_REF][START_REF] Peters | Electrochromism in Photochromic Dithienylcyclopentenes[END_REF][START_REF] Gorodetsky | Bidirectional Ring-Opening and Ring-Closing of Cationic 1,2-Dithienylcyclopentene Molecular Switches Triggered with Light or Electricity[END_REF][START_REF] Moriyama | Electrochemical Cyclization/Cycloreversion Reactions of Diarylethenes[END_REF] Nonetheless, several studies have described the photocyclisation of the DTE moiety through an oxidative process. Indeed, the oxidized open-ring DTE fragment can undergo an internal reorganization affording the closed isomer. [START_REF] Moriyama | Electrochemical Cyclization/Cycloreversion Reactions of Diarylethenes[END_REF][START_REF] Wesenhagen | Photochromism and Electrochemistry of a Dithienylcyclopentene Electroactive Polymer[END_REF][START_REF] Guirado | Understanding Electrochromic Processes Initiated by Dithienylcyclopentene Cation-Radicals[END_REF] Launay et al. [START_REF] Guirado | Understanding Electrochromic Processes Initiated by Dithienylcyclopentene Cation-Radicals[END_REF] have thoroughly studied the electrochemical cyclisation/cycloreversion of DTEs. They suggest that the oxidation of the open form generates a radical cation able to either dimerize or undergo a ring closure to form the closed radical cation through a disproportionation or an ECE mechanism. A few exceptions to the phenomenon have been observed where oxidation leads to the opening of the closed isomer, [START_REF] Peters | Electrochromism in Photochromic Dithienylcyclopentenes[END_REF][START_REF] Gorodetsky | Bidirectional Ring-Opening and Ring-Closing of Cationic 1,2-Dithienylcyclopentene Molecular Switches Triggered with Light or Electricity[END_REF][START_REF] Kleinwächter | Oxidative and Reductive Cyclization in Stiff Dithienylethenes[END_REF][START_REF] Léaustic | Photochromic and Reductive Electrochemical Switching of a Dithiazolylethene with Large Redox Modulation[END_REF] some of these being related to a disproportionation process. [START_REF] Guirado | Understanding Electrochromic Processes Initiated by Dithienylcyclopentene Cation-Radicals[END_REF][START_REF] Areephong | Photo-and Electro-Chromism of Diarylethene Modified ITO Electrodes-towards Molecular Based Read-Write-Erase Information Storage[END_REF][START_REF] Browne | Electro-and Photochemical Switching of Dithienylethene Self-Assembled Monolayers on Gold Electrodes[END_REF] Feringa and Brown also rely on the disproportionation phenomenon to explain the opening reaction of a DTE derivative on a surface once the cationic closed DTE is formed. In this case, two DTE cationic molecules combine affording one in a high oxidation state and another in a low oxidation state, before undergoing an internal reorganization. The same authors also described the disproportionation processes in other electrochemical mechanisms in solution whenever a closed cationic DTE is present. [START_REF] Browne | Oxidative Electrochemical Switching in Dithienylcyclopentenes , Part 2 : Effect of Substitution and Asymmetry on the Efficiency and Direction of Molecular Switching and Redox Stability[END_REF][START_REF] Browne | Oxidative Electrochemical Switching in Dithienylcyclopentenes , Part 1 : Effect of Electronic Perturbation on the Efficiency and Direction of Molecular Switching[END_REF] Taking advantage of the possible disproportionation reaction of the cationic DTE derivatives, our approach is based on the redox processes between the thermodynamically unfavorable generated compounds and the substrate, in order to trap the highly reductive species thus shifting the equilibrium of such a reaction. This constitutes a novel approach to a typical light-promoted redox process, as it takes advantage of both the electro-and photochromic properties of DTEs.

For this purpose, a well-known DTE derivative bearing two pyridiniums entities [START_REF] Peters | Electrochromism in Photochromic Dithienylcyclopentenes[END_REF][START_REF] Gilat | Light-Triggered Molecular Devices : Photochemical Switching of Optical and Electrochemical Properties in Molecular Wire Type Diarylethene Species[END_REF] has been considered as its cationic nature provides unusual redox and optical properties (1 2+ , Scheme 1). Indeed, both isomers present a specific spectroscopic signature that allows to follow the photo-conversion processes between the open and closed forms using absorption spectroscopy (Figure S1). Moreover, the 1c 2+ species can be considered as an oxidative or a reductive species due to its electrochemical behavior characterized by its two redox processes at -0.52 V (1c 2+ /1c + ) and at +1.05 V (1c 3+• /1c 2+ ) vs. Ag + (10 -2 M)|Ag, respectively (Figure S5). The irreversibility of the oxidation wave in the cyclic voltammogram of the closed-ring pyridinium DTE is due to the rapid ring-opening of the oxidized products. [START_REF] Gorodetsky | Bidirectional Ring-Opening and Ring-Closing of Cationic 1,2-Dithienylcyclopentene Molecular Switches Triggered with Light or Electricity[END_REF] The large difference between the redox potentials makes the disproportionation processes thermodynamically unfavorable (Kd=e -E/25.69 ). [START_REF] Richardson | Determination of E2° -E1° in Multistep Charge Transfer by Stationary-Electrode Pulse and Cyclic Voltammetry: Application to Binuclear Ruthenium Ammines[END_REF] However, if an equilibrium exists, as postulated by Le Chatellier's principle, the consumption of one of the products of the reaction will shift the equilibrium in favor of the disproportionation reaction. In that case any molecule for which the oxidation redox potential is located in the range of +1.05 V/-0.52 V should be able to be reduced by 1c + , favoring the disproportionation reaction as a consequence.

Schema 1. Photoisomerization process of 1o 2+ / 1c 2+ couple.

In order to illustrate this phenomenon, the light-triggered reduction of iron (III) bisterpyridine ([Fe(tpy)2] 3+ ) in the presence of the photochromic core 1 2+ is followed using absorption spectroscopy. Indeed, [Fe(tpy)2] 2+ has interesting optical and electrochemical properties: 32 a) a very intense MLCT band (extinction coefficient ~12000 L.mol -1 .cm -1 ) located at 550 nm (Figure S1b), b) the Fe III /Fe II redox potential is located at +0.75 V vs. Ag/Ag + (Figure S4), well in the range for testing the photoreducing character of the considered DTE derivative.

The experiment proceeded as follows: a millimolar solution of [Fe(tpy)2] 2+ was quantitatively oxidized at +0.9 V (1e -/molecule) and the evolution of its UV-vis absorption spectrum was recorded during the electrolysis. The initially deep purple solution, characterized by one main absorption band in the visible region at 550 nm, was bleached during the electrochemical process to give a light-yellow solution. The UV-vis spectrum (Figure S1b In order to further investigate the mechanism of this reaction, an equimolar amount of 1c 2+ solution was added to a [Fe(tpy)2] 3+ solution in the dark. As the 1c 2+ solution was added, the ringopening reaction of the photochromic molecule is evidenced by the decrease of the absorbance band at 650 nm attributed to 1c 2+ (Figure 2). In parallel, the reduction of the [Fe(tpy)2] 3+ to [Fe(tpy)2] 2+ can be observed with the increase of the MLCT absorption band at 550 nm. The latter stops evolving when all 1c 2+ has been consumed, generating a stationary state. Three clear isosbestic points are observed at 379, 452 and 566 nm confirming a confirming a strong interaction between the photochrom and metal complex (Figure 2 andS2). If the solution is then irradiated at 366 nm, the regeneration of the closed form 1c 2+ enables the reduction of the remaining [Fe(tpy)2] 3+ (Figure S2). Based on these experimental data and elements from the literature, [START_REF] Guirado | Understanding Electrochromic Processes Initiated by Dithienylcyclopentene Cation-Radicals[END_REF][START_REF] Areephong | Photo-and Electro-Chromism of Diarylethene Modified ITO Electrodes-towards Molecular Based Read-Write-Erase Information Storage[END_REF][START_REF] Browne | Electro-and Photochemical Switching of Dithienylethene Self-Assembled Monolayers on Gold Electrodes[END_REF] the proposed mechanism for a stochiometric a) b) reaction, can be described as follows: upon irradiation of the solution of [Fe(tpy)2] 3+ in the presence of 1o 2+ , the latter isomerizes to 1c 2+ . A disproportionation process can then occur: (i) two molecules of the dication 1c 2+ leading to the monocation 1c + and the radical trication 1c 3+. , (ii) a redox reaction between the reduced form and Fe(III), affording Fe(II) and 1c 2+ . (iii) once the radical cation 1c 3+. is produced, it isomerizes to 1o 3+. , (iv) which then subsequently oxidizes from another equivalent of 1c 2+ , and regenerates the original closed radical trication and the open dicationic species 1o 2+ . As a consequence of the continuous irradiation, 1o 2+ isomerizes to the closed form and resets the system (v).

The photo-triggered reduction was performed on another substrate featuring several redox processes in order to test its possible generalization. A nickel bis(benzoquinonediimine) complex, 2, showing 3 reversible redox waves and characteristic UV-vis spectra has been studied in presence of 1o 2+ (Figure S3). The irradiation of a 2 2+ solution, obtained after a chemical oxidation with AgSbF6, [START_REF] Ciccione | Geometric and Electronic Structures of Nickel ( II ) Complexes of Redox Noninnocent Tetradentate Phenylenediamine Ligands[END_REF] in the presence of a substoichiometric amount of 1o 2+ (7 %) leads to the quantitative formation of 2 + (E1/2(2 2+ /2 + )=0 V) (Figure 4). The characteristic bands corresponding to the dicationic molecule 2 2+ (512 and 769 nm) disappear with the emergence of bands located at 569 nm and in the NIR at 904 nm, characteristic of the monocationic molecule 2 + . The second reduction (E1/2(2 + /2)=-0.23 V) is not observed probably because this process overlaps with the reduction of 1c 2+ . The substoichiometric nature of the reaction tends to indicate a possible catalytic process. In order to gain further insight into the photocatalytic process, the first reaction was performed and followed varying the number of equivalents (0, 0.1, 0.3, 0.5, 0.7, 1) of 1o 2+ with respect to the iron complex (figure 5). Time dependence was observed with the number of equivalents. For 1 equivalent of 1o 2+ present in solution, a quantitative conversion is obtained in less than 30 min, whereas one hour is needed in the case of 0.5 equivalents. When the irradiation with UV light is performed with less than 0.5 equivalents of 1o 2+ , only a partial reduction of [Fe(tpy)2] 3+ is observed. After one hour, a conversion of 56 and 80% of the iron reduction is reached in the presence of 0.1 and 0.3 equivalents of 1o 2+ , respectively. However, when at least 0.5 equivalents of the photochromic molecule are added to the solution a complete reduction of the [Fe(tpy)2] 3+ to [Fe(tpy)2] 2+ is observed. This experiment confirms that the reduction of [Fe(tpy)2] 3+ by 1o 2+ is a catalytic reaction, as 1c 2+ can be regenerated under continuous irradiation. The photocatalytic cycle is summarized in the scheme 2. The first step of the cycle starts from the photoisomerization of 1o 2+ into 1c 2+ . The presence of Fe (III) shifts the equilibrium of the reaction (i) towards the disproportionation reaction products (1c + and 1c 3+. ). Consequently, Fe(III) is reduced by 1c + to afford Fe (II) and 1c 2+ . In the meantime, 1c 3+. is converted into 1o 3+. . [START_REF] Gorodetsky | Bidirectional Ring-Opening and Ring-Closing of Cationic 1,2-Dithienylcyclopentene Molecular Switches Triggered with Light or Electricity[END_REF] Finally, the presence of 1o 3+. and 1c 2+ in solution leads to a redox reaction regenerating 1c 3+. and the starting reactant 1o 2+ . It should be pointed out that this catalytic behavior can be explained by the presence of an excess of iron (III).

Scheme 2. Photocatalytic cycle of 1o 2+ in presence of an excess of Fe (III).

In conclusion, we have shown for the first time the photo-reducing character of pyridinium substituted DTE derivatives. This reaction occurs through a catalytic mechanism based on a continuous regeneration of the initial 1o 2+ state. It should be noted that other examples of photochromic materials able to induce catalytic reactions are still scarce and present quite modest quantum yields while in this case the reaction is quantitative. This result constitutes a real breakthrough in the field of photo-catalysis.

  ) shows the disappearance of the visible MLCT band attributed to [Fe(tpy)2] 2+ and the shift of the UV-region bands from 333 nm to 353 nm. This spectro-electrochemical experiment confirms a quantitative oxidation of [Fe(tpy)2] 2+ to [Fe(tpy)2] 3+ in solution. At this stage, the addition of one equivalent of 1o 2+ showed an increase in intensity of the bands located in the UV region and attributed to the open form of our molecule. Then, the solution containing a mixture of [Fe(tpy)2] 3+ and 1o 2+ was irradiated with UV light and the absorption spectral changes were recorded. Instead of obtaining the sum of the absorption spectra of the expected closed form 1c 2+ and [Fe(tpy)2] 3+ , the spectral signature of the reduced iron (II) species emerged. During the irradiation time, the absorption peak at 550 nm ascribed to the MLCT band of the ferrous complex increases as well as the band at 650 nm attributed to the closed form of the DTE and reach their maximum, indicating that the photo-induced reduction of [Fe(tpy)2] 3+ occurs by action of the 1 2+ photochromic couple (Figure 1).

Figure 1 .

 1 Figure 1. Evolution of UV-vis absorption spectra during the irradiation (366 nm) of a solution containing 1o 2+ and [Fe(tpy)2] 3+ in acetonitrile at 0.1mM.

Figure 2 .

 2 Figure 2. Evolution of UV-vis absorption spectra a) after addition of 1 equivalent of 1c 2+ to a 0.1 mM solution of [Fe(tpy)2] 3+ (CH3CN + 0.1 M TBAP)

Figure 3

 3 represents the evolution of the band attributed to 1c 2+ and [Fe(tpy)2] 3+ during the experiment. It can be clearly concluded that two equivalents of 1c 2+ are necessary to reduce one equivalent of [Fe(tpy)2] 3+ . Moreover, this experiment shows that the reductive character of 1c 2+ can be triggered by light to reduce [Fe(tpy)2] 3+ as a substrate.

Figure 3 .

 3 Figure 3. Evolution of the absorption intensity at 650nm (a) and 550 nm (b) of a 0.1 mM solution of [Fe(tpy)2] 3+ (CH3CN + 0.1 M TBAP). At t=0 s 1c 2+ is added to the solution, at t=300 s light is switched on (366 nm).

Figure 4 .

 4 Figure 4. Schematics of the photo-reduction process of 2 2+ to 2 + . UV-vis spectra of 2 2+ (plain line) and 2 + (dashed line) in presence of irradiated 1o 2+ (366 nm) in CH2Cl2. [C=0.03mM of 2 2+ and 0.002 mM of 1o 2+ ].

Figure 5 .

 5 Figure 5. Photo-reduction of [Fe(tpy)2] 3+ followed by UV-vis spectroscopy at 8 min (open square), 15 min (full square), 30 min (open circle), 60 min (full circle) irradiation time induced by 0%, 10%, 30%, 50%, 70%, 100% of 1o 2+ . The amount of the reduced form is determined by the absorbance at 550 nm. [C]= 0.1 mM of [Fe(tpy)2] 3+ in CH3CN.

Figure 6 .

 6 Figure 6. Photoreduction of [Fe(tpy)2] 3+ followed by UV/Vis spectroscopy at 8 min (open square), 15 min (full square), 30 min (open circle), 60 min (full circle) irradiation time induced by 0%, 10%, 30%, 50%, 70%, 100% of 1o2+C The amount of the reduced form is determined by the absorbance at 550 nm. [[Fe(tpy)2] 3+ ] = 0.1 mm in CH3CN.

Acknowledgements

The authors wish to acknowledge the support the Institut Universitaire de France.