A Charles, mon directeur de thèse, pour m'avoir proposé ce sujet, encadré et aidé. Merci pour ton soutient, ta disponibilité, et toutes tes idées tout au long de cette année, cette thèse te doit beaucoup.

Aux membres du jury, Monsieur le Professeur Christophe Ribuot, merci d'avoir accepté de présider le jury et de juger ce travail.

Monsieur le Professeur Jean-Luc Cracowski, merci de m'avoir accueillie au sein du CIC, me permettant de réaliser le master puis ma thèse. Votre bienveillance restera pour moi liée à l'ambiance

A ma Mamie qui a préparé cette thèse à mes côtés.

Au début de mes études tu révisais la botanique en P1 avec moi.

A mon Papy qui me manque tellement.

A mes parents, qui m'ont toujours soutenue, tout en veillant sur moi. Pour avoir fait de nous durant toutes ces années, votre priorité. Pour ma Maman, tu es un rayon de soleil tous les jours dans ma vie. Pour mon Papounet que je suis heureuse de rendre fier.

A ma petite soeur, ma Choupie que j'adore. Personne ne sait mieux faire ces grimaces que nous ramenons du bout du monde. Merci de croire en moi envers et contre tout.

A Bruno et Guilaine, pour votre attention, tous vos conseils, votre accueil depuis des années, qui m'ont permis de mûrir. A JC et ses judicieuses remarques au fil des années.

A mon parrain, qui m'a montré l'intérêt des professions de santé, puis soutenu durant mes études.

A mes amies de fac, Nathalie pour tous ces moments partagés et tous ceux à venir ; Audrey qui a assez de folie pour me suivre dans mes rêves, même en ski de fond ; à Mathilde qui la première m'a tendu la main, et depuis en m'a jamais lâché ; à ma Mumu, Adelaïde, Clémence, Alice, Dounia, Salma.

A mes copines de l'internat : Maelle et Elisa qui m'ont accueillies les premières à mon arrivée dans la région, la bonne humeur de Monia, les conseils de Debby, le modèle qu'est Dodo, la douceur de Laura, Lisa et Manon, Héloïse, Julien, Théo et ces idées que nous partageons. A ces 6 mois de coloc avec Karima, un excellent souvenir, grâce à toi. A cette année de master qui m'a permis de rencontrer Marjorie, Lyon et Dijon n'étaient que le début. Pour obtenir un niveau de preuve conséquent, nous avons choisi d'inclure que les essais contrôlés, randomisés, en double aveugle. Nos résultats étant le reflet de la qualité des essais cliniques inclus dans notre analyse, nous avons porté une attention particulière sur la qualité de chaque étude comme sur la qualité de l'analyse globale.

Index des figures

L'ensemble de ce travail est présenté dans le document ci-joint qui sera soumis à publication.

COMPARAISON OF NUTRITIONNAL STRATEGIES TARGETING THE NITRIC OXYDE PATHWAY TO IMPROVE MACROVASCULAR FUNCTION: A SYSTEMATIC REVIEW AND A META ANALYSIS Introduction

Nowadays the interest of the whole population for non-drug approaches to improve their health in rapidly growing. This leaded to the development of a multitude of approaches, from way of life measures to dietary supplementations, botanical other natural products.

Several lifestyle modifications and dietary regimens have showed a positive impact on the onset and severity of chronic diseases and mortality [START_REF] Bloomfield | Effects on Health Outcomes of a Mediterranean Diet With No Restriction on Fat Intake: A Systematic Review and Meta-analysis[END_REF]. One of the most popular measures is dietary supplementation and changes in dietary behavior. The market quickly expanded, promoted by plenty of suppliers carrying plethora of therapeutics benefits.

One of the most widely targeted physiological signaling pathway by dietary supplements is the nitric oxide (NO) pathway. Increasing NO bioavailability has been effective to treat several diseases such as pulmonary arterial hypertension (PAH), heart failure [START_REF] Lundberg | Strategies to increase nitric oxide signalling in cardiovascular disease[END_REF][START_REF] Farah | Nitric oxide signalling in cardiovascular health and disease[END_REF]. The main therapeutic effect is mediated by the improvement of endothelial vascular function, thus leading to a cardiovascular mortality and morbidity reduction. Nitric oxide is produced by nitric oxide synthetases (NOS) from L-arginine, diffuses to the underlying smooth muscle cells and, through soluble guanylate cyclase (sGC), induce the production of cyclic guanosine monophosphate (cGMP), leading to vasodilatation. This pathway has been shown to be down-regulated in several diseases [START_REF] Lundberg | Strategies to increase nitric oxide signalling in cardiovascular disease[END_REF][START_REF] Arnal | Endothelium-derived nitric oxide and vascular physiology and pathology[END_REF][START_REF] Cannon | Role of nitric oxide in cardiovascular disease: focus on the endothelium[END_REF]. Beyond drugs, several nutritional and pharmaco-nutritional approaches have shown the ability to modulate this pathway. Notably, two NOS substrates, L-arginine and its precursor L-citrulline, which is found in large amount in water melon, have been studied. The NOS activity may be also improved by folates in green leafy vegetable, legumes, beans, mushrooms, yeast, some fruits and liver [START_REF] Yuyun | Endothelial dysfunction, endothelial nitric oxide bioavailability, tetrahydrobiopterin, and 5-methyltetrahydrofolate in cardiovascular disease. Where are we with therapy?[END_REF][START_REF] Stanhewicz | Role of folic acid in nitric oxide bioavailability and vascular endothelial function[END_REF], or like synthetic folic acids; through the increase of tetrahydrobiopterin (BH4) recycling, a NOS cofactor [START_REF] Farah | Nitric oxide signalling in cardiovascular health and disease[END_REF]. Furthermore, owning to antioxidant properties, folic acids may prevent NOS uncoupling [START_REF] Farah | Nitric oxide signalling in cardiovascular health and disease[END_REF]. Polyphenols, a large chemistry family of compounds, are contained in fruits and vegetables (e.g. grape and red wine, cocoa, colored berries, onions, tea, soy, citrus…) [START_REF] Burkholder-Cooley | Comparison of polyphenol intakes according to distinct dietary patterns and food sources in the Adventist Health Study-2 cohort[END_REF] and are believed to directly interact with several enzymes involved in the NO pathway [START_REF] Santhakumar | Dietary polyphenols: Structures, bioavailability and protective effects against atherosclerosis[END_REF].

NO can also be produced from nitrate and nitrite by commensal bacteria from the gastrointestinal tract [START_REF] Lundberg | Strategies to increase nitric oxide signalling in cardiovascular disease[END_REF][START_REF] Farah | Nitric oxide signalling in cardiovascular health and disease[END_REF]. Lastly, inorganic nitrate (and nitrite) are contained in food, especially vegetables or beetroot, and NO release compounds like sodium or potassium salt could also be used [START_REF] Hord | Food sources of nitrates and nitrites: the physiologic context for potential health benefits[END_REF].

Whereas pharmaceutical drugs are used to treat diseases, such dietary supplementations are mainly used by healthy people to improve several conditions claimed by manufacturers (e.g. exercise, cardiovascular …) To appraise and compare the efficiency of nutritional or pharmaco-nutritional strategies to increase the NO bioavailability on healthy participants, we performed a systematic review and a meta-analysis. We therefore included randomized clinical trials (RCTs) assessing the efficacy of such interventions on flow-mediated dilatation (FMD), a non-invasive vascular test highly correlated to NO bioavailability [START_REF] Flammer | The Assessment of Endothelial Function -From Research into Clinical Practice[END_REF].

Methods

The meta-analysis was conducted following a predefined protocol (registered on PROSPERO as CRD42018090965) and followed the preferred reporting items for systematic reviews and meta-analyses (PRISMA) for the reporting of our study [START_REF] Moher | Preferred reporting items for systematic reviews and meta-analyses: the PRISMA statement[END_REF].

Interventions and search strategy

We searched Medline and clinicaltrial.gov up to July 4th, 2018 with the following evaluated strategies (nitrite, nitrate, citrulline, arginine, polyphenol, flavonol, resveratrol, cocoa, catechin, coffee, folic acid, hydrogen sulfide) and flow mediated-dilatation (FMD).

We selected the following strategies to modulate the NO pathway:

 NOS substrates and cofactors: L-arginine, L-citrulline and water melon, tetrahydrobiopterin, and separately submitted folic acids.

 NO donors sub-group, we selected inorganic nitrates and nitrites contained in food or in compounds like sodium [START_REF] Heiss | Dietary inorganic nitrate mobilizes circulating angiogenic cells[END_REF][START_REF] Rodriguez-Mateos | Interactions between cocoa flavanols and inorganic nitrate: Additive effects on endothelial function at achievable dietary amounts[END_REF] or potassium [START_REF] Kapil | Inorganic Nitrate Supplementation Lowers Blood Pressure in Humans: Role for Nitrite-Derived NO[END_REF][START_REF] Bahra | Inorganic nitrate ingestion improves vascular compliance but does not alter flow-mediated dilatation in healthy volunteers[END_REF] salt. We restricted the dietary nitrate to vegetables with a very high nitrate content (>250 mg/ 100 g fresh weight): beetroot, celery, cress, chervil, lettuce, spinach, rocket (rucola) [START_REF] Hord | Food sources of nitrates and nitrites: the physiologic context for potential health benefits[END_REF].

 Polyphenol: coming from extracts or dietary ingredient. Then results were classified in sub-groups using Phenol explorer database [START_REF] Neveu | Phenol-Explorer: an online comprehensive database on polyphenol contents in foods[END_REF][START_REF] Pérez-Jiménez | Identification of the 100 richest dietary sources of polyphenols: an application of the Phenol-Explorer database[END_REF][START_REF]Database on Polyphenol Content in Foods -Phenol-Explorer[END_REF]. We used keywords searches general (polyphenol, flavonol) and some products that we well-knew to provide polyphenol (i.e., cocoa, resveratrol, catechin and coffee).

 Hydrogen sulfide: and precursor allicin coming from garlic (4) Only English and French articles were included.

Eligibility criteria

We included only double-blind, randomized, controlled trials (RCT), assessing the efficacy of one of the above-mentioned strategies versus placebo. Moreover, only RCT on healthy adults were included, so without co-morbidities (e.g. hypertension, diabetes, cancer, any cardio-vascular disease). Post-menopause status and smoking was not considered as co-morbidity. Heterogeneous groups containing healthy and non-healthy participants were included only if the results of a sub-group analysis on healthy volunteers were available.

In this meta-analysis we paid special attention to the placebo arms. Indeed, in dietary trials, producing a suitable placebo with a similar taste, color and galenic form should be challenging. Frequently, such placebo was obtained from the studied dietary ingredient depleted of the active substance. However, the processes of depletion are seldom total.

Therefore, most of placebo presented as depleted products contained a small amount of active substance. We considered a depleted product like a placebo if it contained less than 10% of the substance contained into the active arm.

Data extraction

One reviewer (MBG) screened titles and abstracts for inclusion, then reviewed the full-text of potentially relevant articles to determine inclusion using a standardized form according to predefined criteria. The same reviewer (MBG) performed the data extraction on the included full text articles. Other reviewers (JLC, MR, CK) checked the included studies and the extracted data. Discrepancies were resolved through discussion with the team.

The following data were extracted: year, sample size, methodology, participant demographics and baseline characteristics (age, percentage of women, percentage of menopause women, smoker, BMI, cholesterol, LDL, HDL, triglyceride, co-treatment use), follow-up period, wash-out period, details of the intervention and control conditions (type of nutritional or pharmacologic strategy, dosage and treatment duration), outcome (vascular test: FMD, NMD and results), funding.

Risk of bias assessment

We assessed the risk of bias of the included studies according to Cochrane risk of bias tool [START_REF] Pérez-Jiménez | Identification of the 100 richest dietary sources of polyphenols: an application of the Phenol-Explorer database[END_REF]. Finally, we rated their quality as low, medium or high for the following items: random sequence generation; allocation sequence concealment; blinding (participants, personnel); blinding of outcome assessment; completeness of outcome data, selective outcome reporting; and other sources of bias. An item "supported by industrial companies" was added to explain GRADE rating, involving funding, supplementation providing or conflict of interest. We added the overall risk of bias for each trial defined as low-risk if more or equal than 5 high-risk criteria were met, medium-risk between 3 and 4 and low-risk with 2 or less high-risk criteria were met. Then we calculated a quality score (number of low risk items per study) to weighting each study in a sensitivity analysis.

Moreover, we appraised the quality of each therapeutic class using the Grading of Recommendation Assessment, Development and Evaluation (GRADE) recommendations for meta-analysis [START_REF] Guyatt | GRADE guidelines: 4. Rating the quality of evidence-study limitations (risk of bias)[END_REF][START_REF] Guyatt | GRADE guidelines 6. Rating the quality of evidence-imprecision[END_REF][START_REF] Guyatt | GRADE guidelines: 8. Rating the quality of evidence-indirectness[END_REF][START_REF] Guyatt | GRADE guidelines: 7. Rating the quality of evidence-inconsistency[END_REF][START_REF] Guyatt | GRADE guidelines: 5. Rating the quality of evidence-publication bias[END_REF][START_REF] Guyatt | GRADE: an emerging consensus on rating quality of evidence and strength of recommendations[END_REF]. Finally, confidence in quality of results was summarized as very low, low, moderate or high. Details on GRADE evaluation are available in Supplementary

Material

Statistical analysis

We performed two separate meta-analyses to assess the effect of acute (<24h) and chronic supplementations (>24h) of nutritional strategies.

The primary outcome was the mean difference in endothelial vascular function assessed by flow-mediated dilatation of brachial or radial artery after the intervention and control arm. The secondary outcome was change in endothelial-independent vascular function Nitrate-mediated dilatation (NMD). If several FMD were performed in a trial, we extracted the results of FMD on the time-point defined in the primary outcome; if no time point was defined we selected the peak FMD in both arms. When several dosages of the same intervention were tested against a control arm, the results were pooled in a single arm. If a trial presented results for acute and chronic modalities, it was included in both analyses.

A direct meta-analysis using inverse variance fixed effect model or DerSimonian and Laird random effect model was first performed to assess pooled mean difference (MD) and 95% confidence interval of flow-mediated dilatation between groups. Random effect model was used when substantial heterogeneity was detected, I² statistic>50% or p value of the Q statistics < 0.10 indicating heterogeneity. If more than 10 studies were available, the funnel plot asymmetry was explored using Egger's regression test as recommended by Cochrane handbook for systemic reviews [START_REF]Cochrane Handbook for Systematic Reviews of Interventions[END_REF], with a p-value <0.05 suggesting publication bias. All statistical analyses were performed using R statistical software (version 3.2.4).

We secondly performed a Trial Sequential Analysis (beta 0.9.5.10) to adjust the threshold for statistical significance (O'brien and Flemming method) on the available information for each therapeutic class and estimation of required information size [START_REF]Cochrane Handbook for Systematic Reviews of Interventions[END_REF][START_REF] Thorlund | Can trial sequential monitoring boundaries reduce spurious inferences from meta-analyses?[END_REF].

Subgroup and sensitivity analyses

We performed a pre-planned sensitivity analysis excluding high risk of bias studies. Moreover, we assessed the effect of control arms on FMD depending if the control intervention was a placebo or a low dose active substance. We therefore performed a subgroup meta-analysis of pre-post mean differences in the control arms (using a correlation of 0.5).

Several meta-regressions were also performed to assess the potential influence of gender, age, body mass index, total cholesterol, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, triglycerides, proportion of active smokers, FMD before intervention, funding and duration of supplementation (only for chronic intake) on metaanalysis.

Results

Studies selection

1033 references were identified through database searching. After removal of duplicates, titles and abstracts screening, 98 reports proved potential eligibility. Finally, 55 RCTs were included in the quantitative synthesis after full text screening as present in Figure 1. We also performed a GRADE evaluation for each subgroup quality of evidence. Results are provided on Supplementary material.

Acute effect of nutritional strategies

Characteristics of studies and patients

Analysis of the acute effect of selected strategies was conducted on [START_REF] Guyatt | GRADE guidelines: 7. Rating the quality of evidence-inconsistency[END_REF] (2 studies), 3 testing tea extract (2 black and 1green tea), epicatechin (1 study) [START_REF] Ward | Acute effects of chlorogenic acids on endothelial function and blood pressure in healthy men and women[END_REF]. All of them are flavonoids active compound, but we also included 2 studies with chlorogenic acids (i.e. other polyphenol non-flavonoid) [START_REF] Ward | Acute effects of chlorogenic acids on endothelial function and blood pressure in healthy men and women[END_REF][START_REF] Mills | Mediation of coffee-induced improvements in human vascular function by chlorogenic acids and its metabolites: Two randomized, controlled, crossover intervention trials[END_REF]. No study on acute effect of tetrahydrobiopterin, folic acids and hydrogen sulfide met eligibility criteria.

Primary outcome

Overall efficacy of inorganic nitrates on FMD was significant, mean difference (MD) =1.07 (95% CI 0.68, 1.45, I²=47%). Among inorganic nitrate, nitrate salts MD=1.07 (95% CI 0.64-1.49, I²=0%) displayed the larger effect size (Figure 4). Although requested information size is not reach for acute NO donors effect, z-curve showed a positive effect of NO donors in acute effect on the adjusted threshold (Figure 5). In the NOS substrate group, no result was significant, and no trend was apparent (Figure 6). Because less of 5% of requested information was available, the TSA was not performed. For polyphenol analysis, overall efficacy was significant with a MD= 1.45 (95% CI 0.84-2.05, I²=0%) Figure 7. Among this subgroup, overall effect was mainly driven by cocoa products (MD= 2.43, 95% CI 2.45-3.4, I²=89%). In both length of supplementation, polyphenol effect was positive and significant and the requested information size was reached, thus improving the confidence on these results (Figure 8). NOS precursors and NO donors sub-groups funnel plots were not explored because of a too low number of studies. In the polyphenol sub-group, two outliers were visually detected (Figure 9) but the Egger test for funnel plot asymmetry was not significant (p-value= 0.05476).

29

Meta-regressions

Meta-regression showed no influence of the studied covariate except for smoking (pvalue = 0.0357), which was significantly associated to a lower effect of nutritional strategies (Table on supplementary material).

Chronic effect of nutritional strategies

Characteristics of studies and patients

Data of 38 RCTs were included in the meta-analysis of chronic effect, median supplementation time is 28 (interquartile at 25 and 75%: 14-42) days (from 3 to 365 days), on 1076 volunteers, including 46.8 % of women, 8.8% active smokers, mean age was 48.3 ±14.1 years and mean BMI 23.7 ±2.92 kg.m².

12 RCTs selected only post-menopausal women, and 2 RCTs only on active smokers.

For chronic effect analysis, only 3 RCTs constituted the NO donors sub-group, 2 using sodium nitrate salt [START_REF] Rammos | Dietary Nitrate Reverses Vascular Dysfunction in Older Adults With Moderately Increased Cardiovascular Risk[END_REF][START_REF] Devan | Effects of sodium nitrite supplementation on vascular function and related small metabolite signatures in middle-aged and older adults[END_REF] and 1 beetroot juice [START_REF] Lee | Effects of chronic dietary nitrate supplementation on the hemodynamic response to dynamic exercise[END_REF]. We did not find any RCT using lcitrulline meeting our inclusion criteria, therefore NOS precursor were constituted only by arginine per os (7 RCTs), one of which using sustained-release L-arginine [START_REF] Deveaux | l-Arginine Supplementation Alleviates Postprandial Endothelial Dysfunction When Baseline Fasting Plasma Arginine Concentration Is Low: A Randomized Controlled Trial in Healthy Overweight Adults with Cardiometabolic Risk Factors[END_REF]. In contrast, the polyphenol group was composed of 23 RCTs themselves subdivided in 8 categories. The main category was constituted by cocoa (8 RCTs), followed by isoflavones which are another flavonoid class, coming from soy (6 RCTs). Then, 2 RCTs assessed the effect of wine an grape extract [START_REF] Mierlo Laj Van, Zock | Grape Polyphenols Do Not Affect Vascular Function in Healthy Men[END_REF][START_REF] Barona | Grape Polyphenols Reduce Blood Pressure and Increase Flow-Mediated Vasodilation in Men with Metabolic Syndrome[END_REF]; 2 RCTs of stilbene, a non-flavonoid polyphenol, using only stereoisomer trans-resveratrol 99.9% [START_REF] Van Der Made | Trans-Resveratrol Supplementation and Endothelial Function during the Fasting and Postprandial Phase: A Randomized Placebo-Controlled Trial in Overweight and Slightly Obese Participants[END_REF][START_REF] Wong | Chronic resveratrol consumption improves brachial flow-mediated dilatation in healthy obese adults[END_REF]; 2 RCTs assessing the effect of black tea extract [START_REF]Efficacy of Brooke Bond Black Tea Extract on Flow Mediated Dilation in Indian Males -Full Text View -ClinicalTrials[END_REF][START_REF]Effect of Black Tea on Vascular Function -Full Text View -ClinicalTrials[END_REF]; one RCT on blackcurrant juice [START_REF] Khan | Lowering of oxidative stress improves endothelial function in healthy subjects with habitually low intake of fruit and vegetables: A randomized controlled trial of antioxidant-and polyphenol-rich blackcurrant juice[END_REF], expected to bring anthocyanins, flavanol and flavonols; one on citrus hesperidin which is a flavanone [START_REF] Salden | Randomized clinical trial on the efficacy of hesperidin 2S on validated cardiovascular biomarkers in healthy overweight individuals[END_REF], and one on hawthorn extract [START_REF] Asher | Effect of hawthorn standardized extract on flow mediated dilation in prehypertensive and mildly hypertensive adults: a randomized, controlled cross-over trial[END_REF]. We search folate supplementation, but all of 5 RCTs we included provided non dietary ingredient folic acid supplementation. Any RCT about hydrogen sulfide and BH4 was included because expression of FMD was not in percentage.

Primary outcome

For NO donors sub-group, the mean difference was significant (MD= 0.78, 95%CI: 0.16; 1.4, I²=47%). Polyphenols showed a significant improvement of FMD with a MD= 0.83 (95% CI 0.36-1.3, I²= 77%). Two polyphenol groups: cocoa and isoflavones presented the larger effect sizes.

Adjusted threshold analyses corroborate to a polyphenol effect by a significant positive effect and exceeded requested information size.

Secondary outcome

Data retrieved for chronic effect allowed analysis for arginine (5RCTs), for folic acid (3RCTs) and polyphenol (with 6 RCTs for isoflavones, 2 for cocoa, 1 for black tea) only. No significance or trend was detected for any subgroup (Figure on supplementary material).

Meta-regressions

Only 2 meta-regressions were significant. The supplementation duration which any day added improve FMD by 0.01% (p=0.0381). Moreover industrial funded studies presented inferior results by 1.3 % on FMD compared to academic funded (p=0.003).

Sensitivity analysis

Sensitivity analysis excluding high risk of bias studies was performed. In the acute effect meta-analysis, only the polyphenol sub-group contained high quality studies, all study in NOS substrate and NO donors were rated as low or medium quality. The results were similar when excluding high risk of bias studies (presented on GRADE table supplementary material).

Placebo effect

Overall, acute effect of the control interventions on FMD corroborated with a placebo effect MD= 0.11, 95% CI = 0.06; 0.16, I²=0% with no subgroup difference (p=0.15). Likely explained by low dose placebo (<10%), MD= -0.13, 95%CI= -0.19; -0.7). Whereas total placebo effect was non-significant: MD= -0.04, 95% CI= -0.15; 0.08. (Forest plot on supplementary).No placebo/low dose effect was detected in chronic supplementation nor subgroup difference (p=-0.06; 0.13).

Discussion

We conducted this systematic review and meta-analysis to identify the best nutritional compounds able to improve vascular function in healthy volunteers without history, signs or symptoms of cardio-vascular diseases. Our primary conclusion is the limited number of low risk of bias studies despite an abundant literature and numerous trials performed on this topic.

Then, we highlighted that, among nutritional strategies targeting the NO pathway, polyphenols showed the larger effect size on FMD and the robustness level of evidence.

Inorganics nitrates might also be efficient but the level of evidence was lower on chronic effect and did not allow to conclude with certainty, especially in this pathway where tachyphylaxy is well-described. Contrariwise, increasing NOS substrates (L-arginine) or NOS cofactors (folic acids) seemed not an efficient strategy to improve FMD on heathy individuals.

Despite the low number of selected studies, our results on inorganic nitrates group are in accordance with a previous meta-analyses [START_REF] Lara | Effects of inorganic nitrate and beetroot supplementation on endothelial function: a systematic review and meta-analysis[END_REF]. Proposed as a more appropriated alternative to organic nitrates for vascular modulation, inorganic nitrates have shown a dosedependent beneficial effect on endothelial function, sustained in time with no evidence of tolerance [START_REF] Omar | A comparison of organic and inorganic nitrates/nitrites[END_REF]. Inorganic nitrates can be absorbed from vegetables, drinking water, cured meat and nitrate salt supplementation [START_REF] Hord | Food sources of nitrates and nitrites: the physiologic context for potential health benefits[END_REF]. Nitrates are reduced to nitrites by the oral commensal bacteria (50), then the remaining nitrates and 95% of nitrites are rapidly absorbed into the systemic circulation by the upper gastro-intestinal tract [START_REF] Lovegrove | Role of flavonoids and nitrates in cardiovascular health[END_REF]. Absorbed nitrites are thereafter reduced to NO in the blood stream and tissues by several possible mechanisms, which include iron of haem-proteins, myoglobin, xanthine oxidoreductase (XOR), endothelial nitric oxide synthase (eNOS), aldehyde oxidase, ALDH-2, carbonic anhydrase, protons, ascorbate and polyphenols [START_REF] Omar | A comparison of organic and inorganic nitrates/nitrites[END_REF]. NO could easily diffuse to the close environment, form iron-nitrosyl complex with heam molecules. Such binding activates soluble guanylate cyclase (sGC), which generates cyclic guanosine monophosphate (cGMP), leading to vasodilatation, nerve signaling, mitochondrial biogenesis and angiogenesis (3). This mechanism could explain the protecting effects of inorganic nitrites and nitrates as anti-inflammatory and antioxidant on hypertension, diabetes, ischemia/reperfusion, atherosclerosis and thrombosis [START_REF] Münzel | Inorganic nitrite and nitrate in cardiovascular therapy: A better alternative to organic nitrates as nitric oxide donors?[END_REF]. A key mechanism preventing the sudden effect, toxicity of the vascular effect of inorganic nitrites and nitrates is the entero-salivary circulation. In contrast to organic nitrates, 25% of the inorganic nitrates absorbed are concentrated in salivary gland and re-secreted into the mouth [START_REF] Omar | A comparison of organic and inorganic nitrates/nitrites[END_REF][START_REF] Lovegrove | Role of flavonoids and nitrates in cardiovascular health[END_REF]. Finally, even if this is out of the scope of our study, inorganic nitrates should be particularly interesting in pathologic conditions associated with a dysfunctional NOS, because they can increase NO production in a NOS independent manner [START_REF] Farah | Nitric oxide signalling in cardiovascular health and disease[END_REF][START_REF] Lovegrove | Role of flavonoids and nitrates in cardiovascular health[END_REF].

Accordingly to previous meta-analysis [START_REF] Hooper | Flavonoids, flavonoid-rich foods, and cardiovascular risk: a meta-analysis of randomized controlled trials[END_REF][START_REF] Kay | Relative impact of flavonoid composition, dose and structure on vascular function: A systematic review of randomised controlled trials of flavonoid-rich food products[END_REF][START_REF] Hooper | Effects of chocolate, cocoa, and flavan-3-ols on cardiovascular health: a systematic review and meta-analysis of randomized trials[END_REF], we found an improvement of vascular function in pooled analysis with polyphenols both acutely and chronically with an heterogeneous response depending on polyphenolic constituents. From cocoa to chlorogenic acids, the diversity of included supplementations could explain the heterogeneity I²=81% within this group. Cocoa was the only compound showing an acute and a chronic significant effect improving FMD. Concordantly, the blood pressure lowering effect described with flavonol-rich cocoa might also be mediated by the NO pathway [START_REF] Ried | Effect of cocoa on blood pressure[END_REF]. Trials on soy products only provided data on chronic supplementation with a significant positive impact. Chronic effect of red wine or grape did not show significant efficacy. Polyphenols are well known to improve vascular function, notwithstanding the mechanism of action is not fully understood [START_REF] Lovegrove | Role of flavonoids and nitrates in cardiovascular health[END_REF]. Traditionally described as antioxidant scavenging reactive oxygen species (ROS), their bio-availability about 5 to 10% of total intake, seems too low to support this hypothesis [START_REF] Lundberg | Strategies to increase nitric oxide signalling in cardiovascular disease[END_REF][START_REF] Scalbert | Polyphenols: antioxidants and beyond[END_REF][START_REF] Santhakumar | Dietary polyphenols: Structures, bioavailability and protective effects against atherosclerosis[END_REF]. An additional effect is inhibition of ROS-generating NADPH oxidase, preventing NO destruction by radicals which can interact with. Moreover, polyphenols stimulate eNOS, through phosphorylation, by increasing calcium concentration, PI3 kinase/AKT pathway and p38 mitogen-activated protein kinase [START_REF] Lundberg | Strategies to increase nitric oxide signalling in cardiovascular disease[END_REF][START_REF] Yamagata | Dietary polyphenols regulate endothelial function and prevent cardiovascular disease[END_REF]. Furthermore, other mechanisms are hypothesized : anti-inflammatory (decrease of adhesion molecules [START_REF] Yamagata | Dietary polyphenols regulate endothelial function and prevent cardiovascular disease[END_REF], part by xanthine oxidase modulation [START_REF] Lovegrove | Role of flavonoids and nitrates in cardiovascular health[END_REF], action on inflammatory cell and mediator), decrease of platelet aggregation, improvement of circulating lipid profile reducing atherogenesis [START_REF] Yamagata | Dietary polyphenols regulate endothelial function and prevent cardiovascular disease[END_REF]. Nevertheless, numerous compounds with variable activity are pooled under the "polyphenol" designation. Vascular effect is mostly described for flavonoids but an effect of other polyphenol could not be excluded (stilbene, chlorogenic acid). Given that, one dietary ingredient may contain several polyphenols in different amounts, further complicated by interaction and possible additive effects, to identify the compound inducing the therapeutic effect is challenging.

Moreover, an additive effect was also recently described between nitrites/nitrates and flavonoids [START_REF] Rodriguez-Mateos | Interactions between cocoa flavanols and inorganic nitrate: Additive effects on endothelial function at achievable dietary amounts[END_REF]. As described above, nitrite can be metabolized in NO by eNOS, which can be stimulated by polyphenols. In the stomach, reduction of nitrite to NO is enhanced by the presence of vitamin C or polyphenol (by reductive hydroxyl group on the phenol ring) [START_REF] Lundberg | Strategies to increase nitric oxide signalling in cardiovascular disease[END_REF][START_REF] Lovegrove | Role of flavonoids and nitrates in cardiovascular health[END_REF].

Despite a lack of evidence in tissues, effects of inorganic nitrates and cocoa flavonols could be additive to improve vascular function in healthy humans [START_REF] Rodriguez-Mateos | Interactions between cocoa flavanols and inorganic nitrate: Additive effects on endothelial function at achievable dietary amounts[END_REF]. Furthermore nitration or nitrosation on polyphenols could result in longer NO donors half-life (3). So, the positive effect of inorganic nitrates contained in beetroot should be reconsidered. Indeed polyphenols (flavonoids: luteolin and quercetin and lignans) are present in beetroot, especially in beetroot juice (59) [START_REF] Neveu | Phenol-Explorer: an online comprehensive database on polyphenol contents in foods[END_REF]. Likewise, co-ingestion of methyl xanthines (caffeine or theobromine) in cocoa, coffee and tea, could enhance flavonol absorption leading to a synergic effect on vascular function [START_REF] Sansone | Methylxanthines enhance the effects of cocoa flavanols on cardiovascular function: randomized, double-masked controlled studies[END_REF].

The NOS precursor group was mostly represented by per os L-arginine intervention trials (except one on intravenous arginine and another one on citrulline). This semi-essential amino-acid seemed to be an attractive way of supplementation as a substrate of NOS in epithelial cells, increasing its concentration enhancing NO production. Our results constituted a good reflect of the very inconsistent literature on this topic and are concordant with a previous meta-analysis [START_REF] Bai | Increase in fasting vascular endothelial function after short-term oral l-arginine is effective when baseline flow-mediated dilation is low: a meta-analysis of randomized controlled trials[END_REF]. In their work, L-arginine intake (from 3 to 24 g/d) improved FMD only if baseline FMD was low but has no effect of medium FMD. Thus, L-arginine might restore a dysfunctional endothelium by normalizing impaired NO production.

Nevertheless, normal endothelial function could not be further improved in that way. One way could be to rebalance the L-arginine/Asymmetric dimethyl arginine (ADMA) ratio; given that ADMA is an endogen NOS inhibitor. ADMA is overproduced in cardiovascular disease [START_REF] Böger | The Pharmacodynamics of L-Arginine[END_REF].

In the first instance, their conclusion seems to explain our results, because our population is overall healthy. But, the larger observed effect size was not observed in the lower FMD population had no cardiovascular disease. These two characteristics could explain the nonsignificant tendency of FMD improvement we concluded.

Unfortunately we were not able to assess the efficacy of two identified strategies because of the absence of trials fitting our inclusion criteria. Tetrahydrobiopterin, a NOS cofactor, showed promising results on animals (68) but inconsistent results in clinical studies [START_REF] Farah | Nitric oxide signalling in cardiovascular health and disease[END_REF][START_REF] Yuyun | Endothelial dysfunction, endothelial nitric oxide bioavailability, tetrahydrobiopterin, and 5-methyltetrahydrofolate in cardiovascular disease. Where are we with therapy?[END_REF]. An hypothesis is that BH4 is immediately oxidized in dihydrobiopterin (BH2) (4), so ratio BH4/BH2 remains unchanged even in supplementation group [START_REF] Cunnington | Systemic and Vascular Oxidation Limits the Efficacy of Oral Tetrahydrobiopterin Treatment in Patients With Coronary Artery Disease[END_REF]. Lastly, we planned to study hydrogen sulfide (H2S). This promising compound can be supplied by allicin contained in garlic (4). H2S act on 3 level in NO-sGC pathway, up to reducing oxidative stress by activating eNOS; enhancing nitrites reduction by xanthine oxidoreductase (XOR), leading to a NOS independent production of NO; stimulating sGC activity during oxidative stress and inhibiting PDE5 (4). On the 2 studies selected in our literature review, none were included due to results expression. One of them ( 70), conducted on young, trained and healthy men with acute garlic supplementation, didn't find improvement of FMD. However, 7 of 18 participants were excluded of the analysis, leading to a major suspected bias. In chronic supplementation with aged garlic extracts (aged for better concentration of sulfur), FMD were improved after vascular challenge of methionine compared with placebo [START_REF] Weiss | Significance of Garlic and Its Constituents in Cancer and Cardiovascular Disease[END_REF]. This result cannot be interpreted in regard to our analysis, because vascular challenges were excluded.

Almost all dietary source compositions are variable, complex and partially unknown.

In dietary ingredients, other chemicals are presents. Dietary fiber, vitamins, amino-acids, ions (like nitrite or nitrates) in vegetables or fruits can affect the effect of the studied component.

In addition, the composition of the same type of food could vary depending on growth conditions (soil, temperature, sunlight, water, genotype…) storage, transportation or processing (washing, peeling, cooking) [START_REF] Hord | Food sources of nitrates and nitrites: the physiologic context for potential health benefits[END_REF][START_REF] Yamagata | Dietary polyphenols regulate endothelial function and prevent cardiovascular disease[END_REF]. For example, in vegetable, the level of artery before and after dilatation in millimeter. The result expressions restricted statistical performed of previous meta-analysis on non-far away topic [START_REF] Costello | A select review reporting the quality of studies measuring endothelial dysfunction in randomised diet intervention trials[END_REF]. In contrast, we preferred excluded some RCTs without FMD in percentage, to keep coherent comparison despite information loss.

Although we tried to have a homogenous healthy population, smoking, BMI, lipid profile, sex-ratio, post-menopausal status for women, age, could influence the vascular function. However, only smoking significantly impacted the results on acute effect, according to meta-regression.

Duration of supplementation varied from 3 days to 1 year; median supplementation time was 28 (interquartile range at 25 and 75%: IQR 14-42) days. The meta-regression was significant and corroborate that each added day of supplementation increase FMD by 0.011 % (p-value= 0.0381). In acute administration, a potential bias could be produced by the measurement time of FMD regarding to the plasmatic peak of the studied molecule.

Placebo also differed by form and composition among the included studies, moreover placebo were mixed with low dose substances. In our meta-analyses, placebo increased mean FMD from 0.11% (95% CI 0.06, 0.16) on acute effect and was not significant in chronic supplementation. No difference in subgroup analysis between placebo and low dose compounds was detected. In our study, to be considered as a placebo the composition of the control arm must be composed of less than 10 % active molecule contained in the active arm.

We based this choice on a batching of beetroot juice and nitrate-depleted beetroot juice which nitrate concentration were 3.5 mmol/l and 0.34 mmol/l respectively [START_REF] Notay | Acute beetroot juice supplementation on sympathetic nerve activity: a randomized, double-blind, placebo-controlled proof-of-concept study[END_REF][START_REF] Bondonno | Absence of an effect of high nitrate intake from beetroot juice on blood pressure in treated hypertensive individuals: a randomized controlled trial[END_REF]. Nevertheless, these studies also shown that compound of interest could be lower than reported by the manufacturer. Several studies didn't perform independent chemical analysis or only partially and selectively. We therefore pooled polyphenols by food origin rather than by major active compounds. We agreed that this choice may have biased our analysis because same dietary food can supply different active molecules, leading to a mismatch between substantial intake and sub-group results [START_REF] Kay | Relative impact of flavonoid composition, dose and structure on vascular function: A systematic review of randomised controlled trials of flavonoid-rich food products[END_REF].

A major limitation of our analysis was the quality of included RCTs, although we paid special attention to it. Included studies had to be double blinded, randomized and placebo controlled. Indeed, the overall Cochrane quality score was at maximum of 6 for only 2 studies (33,77) and of 5 in 2 studies (42,78,79) Notwithstanding, the sensitivity analyses excluding high risk of bias studies was consistent with the main analysis. Actually, dissenting opinions are expressed on study quality in nutrition, in particular versus study quality required for drug trials [START_REF] Costello | A select review reporting the quality of studies measuring endothelial dysfunction in randomised diet intervention trials[END_REF][START_REF] Barnard | The Misuse of Meta-analysis in Nutrition Research[END_REF]. However, all of them highlight the importance of quality given that conclusions of nutrition research are highly publicized and impact feeding behavior and policies on diet and health [START_REF] Barnard | The Misuse of Meta-analysis in Nutrition Research[END_REF].

Funding is often quote to impact either results or quality of studies [START_REF] Barnard | The Misuse of Meta-analysis in Nutrition Research[END_REF][START_REF] Mozaffarian | Dietary guidelines and health-is nutrition science up to the task?[END_REF]. However, in chronic supplementation, industry funded studies seem to support a smaller effect size in meta-regression (β= -1.55, p-value=0.0030). In contrast, Chartres and al. [START_REF] Chartres | Association of Industry Sponsorship With Outcomes of Nutrition Studies: A Systematic Review and Meta-analysis[END_REF] depicted a nonsignificant tendency of favorable conclusions in industry sponsored RCTs compared to academic sponsored. Nevertheless, funding sources were not often disclosed [START_REF] Chartres | Association of Industry Sponsorship With Outcomes of Nutrition Studies: A Systematic Review and Meta-analysis[END_REF]. In this meta-analysis, industries sponsored RCTs displayed equal or better quality score, although score do not assess the whole risk of bias [START_REF] Chartres | Association of Industry Sponsorship With Outcomes of Nutrition Studies: A Systematic Review and Meta-analysis[END_REF]. For example, publication bias and restricted publication of negative industrial studies should be suspected for polyphenol in our metaanalysis.

Conclusion

In this study, we compared and assessed the effect of several nutritional strategies targeting the NO pathway on vascular function of healthy volunteers. We found that polyphenols had the larger effect size and the best evidence to support a positive effect on FMD. Moreover, while less robustly, inorganic nitrates also showed a positive effect.

Contrariwise evidence is lacking to support an effect of NOS substrates and folic acid supplementation. More high-quality trials are mandatory to precisely assess the impact of the different strategies acting on the NO pathway, notably tetrahydrobiopterin or hydrogen sulfide. Meanwhile the active substances contained in each dietary supplement should be thoroughly identified as their mechanisms, factoring overall composition and interaction in.

Discussion

Sur

Figure 1 :

 1 Figure 1: Flow chart .................................................................................................................

Figure 2 :

 2 Figure 2: quality of studies on acute supplementation .............................................................

Figure 3 :

 3 Figure 3: quality of studies on chronic supplementation .........................................................

Figure 4 :

 4 Figure 4: Acute effect of NO donors on FMD (Forest plot) ....................................................

Figure 5 :

 5 Figure 5: Z-curve of acute effect of NO donors on FMD ........................................................

Figure 6 :

 6 Figure 6: Acute effect of NOS substrates on FMD (Forest plot) .............................................

Figure 7 :

 7 Figure 7: Acute effect of polyphenol on FMD (Forest plot) ....................................................

Figure 8 :

 8 Figure 8:Z curve acute effect of polyphenols on FMD ............................................................

Figure 9 :

 9 Figure 9: funnel plot of polyphenol acute effect ......................................................................

Figure 10 :

 10 Figure 10: chronic effect of nitrates on FMD (Forest plot) ......................................................

Figure 11 :

 11 Figure 11: Z-curve of chronic NO donors on FMD .................................................................

Figure 12 :

 12 Figure 12: Chronic effect of NOS substrates (Forest plot) ......................................................

Figure 13 :

 13 Figure 13: Z-curve chronic NOS substrates on FMD ..............................................................

Figure 14 :

 14 Figure 14: Chronic effect of folic acids on FMD (Forest plot) ................................................

Figure 15 :

 15 Figure 15: Z-curve on chronic folic acids on FMD .................................................................

Figure 16 :

 16 Figure 16: Chronic effect of polyphenols on FMD (Forest plot) .............................................

Figure 17 :

 17 Figure 17: Z-curve chronic effect of polyphenols on FMD .....................................................

Figure 18 :

 18 Figure 18: Funnel plot chronic effect of polyphenols on FMD ...............................................

Figure 19 :

 19 Figure 19: results summary ......................................................................................................

Figure 20 :

 20 Figure 20: acute effect of NO donors on NMD .......................................................................

Figure 21 :

 21 Figure 21: acute effect of NOS substrates on NMD ................................................................

Figure 22 :

 22 Figure 22: acute effect of polyphenol on NMD .......................................................................

Figure 23 :

 23 Figure 23: chronic effect of NOS substrates on NMD .............................................................

Figure 24 :

 24 Figure 24: chronic effect of folic acids on NMD .....................................................................

Figure 25 :

 25 Figure 25: chronic effect of polyphenols on NMD .................................................................

Figure 26 :

 26 Figure 26: placebo effect on acute supplementation ...............................................................

Figure 27 :

 27 Figure 27: placebo effect on chronic supplementation ............................................................

  Records identified through database search in and screened N(PubMed)=885 N(clinical trial)= 148 Full text articles assessed for eligibility N(Pubmed)= 81 N(clinical trial)= 17 Records excluded N( Pubmed)= 794 N(clinical trial)=131

Figure 2 :

 2 Figure 2: quality of studies on acute supplementation

Figure 4 :

 4 Figure 4: Acute effect of NO donors on FMD (Forest plot)

Figure 5 :

 5 Figure 5: Z-curve of acute effect of NO donors on FMD

Figure 6 :

 6 Figure 6: Acute effect of NOS substrates on FMD (Forest plot)

Figure 7 :

 7 Figure 7: Acute effect of polyphenols on FMD (Forest plot)

Figure 8 :

 8 Figure 8:Z curve acute effect of polyphenols on FMD

Figure 9 :

 9 Figure 9: funnel plot of polyphenols acute effect

Figure 10 :

 10 Figure 10: chronic effect of nitrates on FMD (Forest plot)

Figure 11 :

 11 Figure 11: Z-curve of chronic NO donors on FMD

Figure 12 :

 12 Figure 12: Chronic effect of NOS substrates (Forest plot)

Figure 13 :

 13 Figure 13: Z-curve chronic NOS substrates on FMD

Figure 14 :

 14 Figure 14: Chronic effect of folic acids on FMD (Forest plot)

Figure 16 :

 16 Figure 16: Chronic effect of polyphenols on FMD (Forest plot)

Figure 18 :

 18 Figure 18: Funnel plot chronic effect of polyphenols on FMD

Figure 21 :Figure 23 : 68 Figure 24 :

 21236824 Figure 21: acute effect of NOS substrates on NMD

Figure 26 :

 26 Figure 26: placebo effect on acute supplementation

Table 2

 2 études pour le jus de betterave et 1 pour les épinards rouges. Pourtant ce type de supplémentation est spécialement intéressant car augmente la biodisponibilité de NO de manière indépendante de la NOS puisque les nitrates sont transformés en nitrites par la flore buccale puis en NO par différentes enzymes des cellules endothéliales.Au même niveau de régulation de la voie du NO, il faut considérer ici les stratégies visant à augmenter les cofacteurs de la NOS. Leur importance vient du fait qu'un déficit en substrat ou cofacteur de la NOS entraine son découplage, et donc une production délétère de ROS, plutôt que celle de NO, protectrice de la fonction vasculaire. Pour la BH4 aucune étude n'a été inclue dans notre analyse, la littérature étant restreinte sur ce sujet. Ensuite nous nous sommes intéressés aux folates qui peuvent soit activer la NOS directement par liaison à la place de la BH4, soit augmenter la régénération de la BH4, en plus de leur effet antioxydant.Le résultat pour ces compléments alimentaires (aucun aliment inclus dans ce groupe) est décevant car non significatif, et proche de la futilité selon les courbes de seuil ajusté. La notion de futilité indique qu'une absence d'effet est significativement démontrée.

	1 033 références identifiées, nous avons finalement inclus dans notre analyse 55
	essais cliniques (53 venant de Pub Med et 2 de clinicaltrial.gov) permettant une analyse sur 24
	essais en supplémentation aigue et 38 en chronique.
	Selon notre analyse, les nitrates inorganiques montrent un effet positif et significatif en
	supplémentation aigue comme chronique. Cependant si ce résultat semble confirmé avec les
	seuils ajustés pour la supplémentation aigue, ceci n'est pas le cas pour la supplémentation
	chronique qui est loin de l'effectif informatif (433 patients pour 80 inclus). Il paraît donc
	difficile d'affirmer formellement un effet des nitrates en chronique, en particulier dans cette
	voie pour laquelle une forte tachyphylaxie est déjà décrite pour les médicaments. De plus
	notre analyse porte particulièrement sur les sels sodiques ou potassiques de nitrate (6 études),
	contre seulement 3

Table 3 :

 3 summary of studies included in meta-analysis

			Number
	Studies	Design	of	Supplementation	Group	Dura	wash	Cuff	NMD
			subjects			tion	out
					NOS
	Adams1995	crossover	12 L-arginine PO	substrates	3	7 forearm yes
	Anisha
	Pargal2017	crossover	27 Black tea extract polyphenol	7	6 forearm no
				hawthorn
				standardized
	Asher2012	crossover	21	extract	polyphenol	3	1,5 forearm no
				grape
				polyphenol
	Barona2012	crossover	24	powder (GRAPE) polyphenol	30	21 forearm no
					NOS
	Blum2000	crossover	10 L-arginine PO	substrates	30	30 forearm yes
	Bode-				NOS
	Boger2003	crossover	12 L-arginine PO	substrates	14	14 forearm yes
					NOS
	clarkson1996	crossover	27 L-arginine PO	substrates	28	28 forearm yes
	Colacurci2004	parallel	57 isoflavone	polyphenol	180 NA	arm	yes
	Cuevas2003	crossover	18 soy isoflavone	polyphenol	28	0 forearm yes
	Davison2008	parallel	49 cocoa	polyphenol	84 NA	forearm no
	Decroix2018	crossover	14 cocoa flavonol	polyphenol	6	7 forearm no
	DeVan2016	Parallel	31 nitrite	NO donors	70 NA	MD	no
				sustained-
				release L-	NOS
	Deveaux2016	crossover	33	arginine	substrates	28	28 forearm yes
				dark chocolate
	Engler2004	Parallel	21	bars	polyphenol	14 NA	MD	no
	Grassi2015	crossover	20 cocoa	polyphenol	7	7 forearm no
	Hale2002	Parallel	29 soya isoflavone polyphenol	14 NA	MD	no
	Hallund2006	crossover	28 soya isoflavone polyphenol	56	56 forearm yes
	Heiss A2015	Parallel	20 cocoa flavonol	polyphenol	14 NA	MD	yes
	Heiss B2015	Parallel	20 cocoa flavonol	polyphenol	14 NA	MD	yes

Table 4 :

 4 characteristics of subjects included in meta-analysis

	Studies	% women	% smoker	Age (years)	BMI	Choles terol mmol/l	LDL mmol/l	HDL mmol/l	TG mmol/l
	Adams1995	0	0	34, SD: 3,46	MD	5,43	4,34	1,11	1,4
	Anisha
	Pargal2017	0	0	36, SD: MD	23,2	MD	MD	MD	MD
	Asher2012	57,2	0	50,9, SD: 10,5	29,3	MD	MD	MD	MD
	Barona2012	0	MD	51,3, SD: 9,6	MD	MD	MD	MD	MD
		100 (100 %
		post
	Blum2000	menopause)	0	55, SD: 5	MD	5,14	3,2	1,55	1,09
		33 (100 %
	Bode-	post
	Boger2003	menopause)	0	73,8, SD: MD	MD	MD	MD	MD	MD
	clarkson1996	33	0	29, SD: 5	MD	7,6	6,15	1,29	1,36
		100 (100 %
		post
	Colacurci2004	menopause)	0	55,15, SD: 3,85	25,8	MD	3,65	1,55	1,54
		100 (100 %
		post
	Cuevas2003	menopause)	0	59, SD: MD	29,3	7,39	5,03	1,39	2,14
	Davison2008	64,89	MD	45,12, SD: MD	33,4	5,13	3,49	1,51	1,51
	Decroix2018	0	0	30,7, SD: 3,1	22,5	MD	MD	MD	MD
		45 (100 %
		post
	DeVan2016	menopause)	0	61,71, SD: 7,45	24,6	4,47	2,58	1,49 0,842
	Deveaux2016	27	0	45, SD: 8,9	28	2,1	1,2	0,4	2,4
	Engler2004 47,6428571	0	32,13, SD: 9,93	22,5	4,53	2,29	1,59	1,2
	Grassi2015	45	0	53,8, SD: 8,9	25,4	MD	3,4	1,43	1,29
		100 (100 %
		post
	Hale2002	menopause)	0	57,17, SD: 5,71	24,9	5,05	3,97	1,63	1,21
		100(100 %
		post
	Hallund2006	menopause)	0	57, SD: 5	24	5,98	3,91	1,53	1,18
	Heiss A2015	0	0	26, SD: 4,47	24,9	4,45	3,33	1,37	1,09
	Heiss B2015	0	0	60, SD: 8,94	26,5	5,35	4,06	1,39	1,33
		47 (100 %
	Hermansen20	post
	05	menopause)	39	59,54, SD: 3,89 26,075	6,73	4,41	1,41	MD
		100 (none
		post
	Hoch2009	menaupose)	0	24, SD: 3,21	22,1	MD	MD	MD	MD
	Khan2014	32,98	84,625 52,38, SD: 9,67	28,8	5,2	MD	MD	MD
	Lee2015	0	0	22, SD: 3,74	23,4	MD	MD	MD	MD
	Lorenzo
	Ghiadoni2018	0	0	43, SD: MD	25	MD	MD	MD	MD
	Olthof2006	42,5	MD	59, SD: 5	25,5	5,8	MD	MD	MD

Table 5 :

 5 meta-regression on acute FMD

	Variable	Rate of available	beta	95 CI	P value
		variable
	Women proportion	26	-0.0027	-0.0263 0.0209 0.8223
	Smoker proportion	20	0.0155	0.0010 0.0300	0.0357
	Age	27	-0.0009	-0.0372 0.0353	0.9594
	BMI	24	0.0347	-0.0622 0.1316	0.4827
	Cholesterol	14	-0.2947	-1.7042 1.1149	0.6820
	LDL	9	0.3678	-0.6050 1.3405	0.4587
	HDL	12	0.3763	-4.4218 5.1744	0.8778
	Triglyceride	12	-1.0934	-3.2645 1.0778	0.3236
	FMD before intervention 19	0.0227	-0.5083 0.5536	0.9332
	Funding	40	-0.2338	-1.1557 0.6882	0.6192

Table 6 :

 6 meta-regression on chronic FMD

	Variable	Rate of available	beta	95 CI	P value
		variable
	Women proportion	40	0.0057	-0.0076 0.019	0.40
	Smoker proportion	33	-0.0002	-0.0230 0.0227	0.9884
	Age	40	0.0015	-0.0309 0.0338	0.9294
	BMI	33	-0.0513	-0.1422 0.0396	0.2686
	Cholesterol	25	0.5471	-0.1057 1.1998	0.1005
	LDL	22	0.5204	-0.0707 1.1115	0.0844
	HDL	23	4.3288	-0.4387 9.0963	0.0751
	Triglyceride	22	0.2048	-1.4024 1.8120	0.8028
	FMD before intervention	30	-0.1015	-0.4085 0.2055	0.5169
	Duration of	40	0.0110	0.0006 0.0225	0.0381
	supplementation
	Funding	40	-1.3358	-2.2440 -0.4277	0.0039

Quant au financement, les études industrielles présentent des résultats de taille d'effet inférieur de 1.3 % par rapport aux études académiques. Afin d'apprécier la cohérence de nos résultats en fonction de la qualité des études inclues, des analyses de sensibilité ont été menées en excluant les essais à haut risque de biais.Tous les résultats ainsi obtenus étaient cohérents avec les précédents. Ces analyses, associées au financement et à nos résultats de la méta-analyse principale ont aussi servies à établir le niveau de preuve de nos résultats via le système GRADE. La qualité conclue est donc très faible pour toutes les analyses (nitrates inorganiques, substrats de la NOS et acide folique) en supplémentation chronique sauf pour les polyphénols pour lesquels elle est faible. En supplémentation aigue la qualité estimée est dans l'ensemble meilleure : faible pour les substrats de la NOS et les polyphénols et intermédiaire pour les nitrates inorganiques.

Remerciements

de travail unique qu'est celle du CIC. Madame le Docteur Nathalie Fouilhé Sam-Laï, pour avoir accepté sans hésitation de faire partie du jury de cette thèse. Monsieur le Docteur Jeremy Bellien, merci d'accepter de juger ce travail.

Summary results

The results are summarized in Figure 19: results summary . Constituents on NO pathway are in blue boxes. Light blue ones represent studied supplementations. In two circle above, effect are summarize by mean difference ( confidence intervals at 95 %) for acute and chronic supplementation, color varies according to the significativity (green if result is significant and orange if it is not). For each ones, GRADE quality of evidence assessments are indicated by: intermediate, : low and : very low. group, maybe coincidence because seldom studies. The only consistent findings that Larginine is on the cross way of number physiological processes: precursor of several other amino-acid, part of urea cycle as substrate for arginase, substrate for NOS [START_REF] Lundberg | Strategies to increase nitric oxide signalling in cardiovascular disease[END_REF]. So, in a complex integrated system, with sprawling regulation.

Several NOS co-factors could be increase as tetrahydrobiopterin supplementation had also been considered. Especially because when NOS substrate or cofactor have limited bioavailability, NOS can be uncoupled [START_REF] Förstermann | Therapeutic effect of enhancing endothelial nitric oxide synthase (eNOS) expression and preventing eNOS uncoupling[END_REF], resulting in the production of superoxide radical rather than NO [START_REF] Lundberg | Strategies to increase nitric oxide signalling in cardiovascular disease[END_REF][START_REF] Farah | Nitric oxide signalling in cardiovascular health and disease[END_REF]. Folates also named vitamin B9 are not synthetized de novo by mammals and depend of dietary intake or supplementation. Green leafy vegetable, legumes, beans, mushrooms, yeast, some fruits and liver, provide the majority of nutritional intake [START_REF] Yuyun | Endothelial dysfunction, endothelial nitric oxide bioavailability, tetrahydrobiopterin, and 5-methyltetrahydrofolate in cardiovascular disease. Where are we with therapy?[END_REF][START_REF] Stanhewicz | Role of folic acid in nitric oxide bioavailability and vascular endothelial function[END_REF]. Because bio availability of folates is rather low, around 50%, pharmaco-nutritional supplementation as synthetic folic acids is usual [START_REF] Stanhewicz | Role of folic acid in nitric oxide bioavailability and vascular endothelial function[END_REF]. After a dose dependent absorption (4), folates are bio-activated in 5-methyl tetrahydrofolate (5MTHF) by the liver [START_REF] Yuyun | Endothelial dysfunction, endothelial nitric oxide bioavailability, tetrahydrobiopterin, and 5-methyltetrahydrofolate in cardiovascular disease. Where are we with therapy?[END_REF] ; and if saturated, folate can be reduced in cell by dihydrofolate reductase (DHFR) [START_REF] Stanhewicz | Role of folic acid in nitric oxide bioavailability and vascular endothelial function[END_REF].

Mechanistically, two main actions are described on vascular function, a direct antioxidant effect by ROS scavenging [START_REF] Yuyun | Endothelial dysfunction, endothelial nitric oxide bioavailability, tetrahydrobiopterin, and 5-methyltetrahydrofolate in cardiovascular disease. Where are we with therapy?[END_REF] or an increase of NOS coupling through direct and indirect ways. 5MTHF is presumed to mimic the BH4 conformation and bind the NOS promoting NO production [START_REF] Hyndman | Interaction of 5methyltetrahydrofolate and tetrahydrobiopterin on endothelial function[END_REF]. Moreover, tetrahydrofolate upregulate DHFR activity, recycling BH4 from inactive BH2 [START_REF] Farah | Nitric oxide signalling in cardiovascular health and disease[END_REF][START_REF] Stanhewicz | Role of folic acid in nitric oxide bioavailability and vascular endothelial function[END_REF]. A previous meta-analysis on this topic (65) found a dose dependent improvement of FMD, especially with folic acid supplementation above 5 mg/j. They also conclude to an improvement independent to the homocysteine lowering effect. The FMD improvement impact larger on participants with high cardiovascular risks, consistent with literature (4). Our results were different. Nevertheless, on the 5 studies included in our analysis, one used less than 5 mg/d (0.8 mg/d) (66), and another just 5 mg/d [START_REF] Woodman Rj | Folic acid does not improve endothelial function in healthy hyperhomocysteinaemic subjects[END_REF]. Adding our nitrates are higher if they grew with nitrogen containing fertilizers than in organic vegetables [START_REF] Hord | Food sources of nitrates and nitrites: the physiologic context for potential health benefits[END_REF]. About nitrite/nitrate and polyphenol, the microbiota should also be considered.

Biotransformation of polyphenols by the gut microbiota increase bio-availability of polyphenol (breaking the large structures to cross intestinal barrier) but also increase the diversity in absorbed polyphenols [START_REF] Santhakumar | Dietary polyphenols: Structures, bioavailability and protective effects against atherosclerosis[END_REF]. In a study, the change in microbiome by prebiotics induced an improvement in NO dependent endothelial function [START_REF] Catry | Targeting the gut microbiota with inulin-type fructans: preclinical demonstration of a novel approach in the management of endothelial dysfunction[END_REF].The last point to consider is the importance of supplementation versus the intakes in daily food. For polyphenol, coffee consumer ingest near up to triple amount of polyphenol (caffeic acid) than non-coffee consumer [START_REF] Burkholder-Cooley | Comparison of polyphenol intakes according to distinct dietary patterns and food sources in the Adventist Health Study-2 cohort[END_REF]. Of course, it can lead to a major bias if supplementation intake is too low regarding daily food consumption or in inhomogeneous population. In the meta-analysis on Larginine [START_REF] Bai | Increase in fasting vascular endothelial function after short-term oral l-arginine is effective when baseline flow-mediated dilation is low: a meta-analysis of randomized controlled trials[END_REF], in front of the same efficacy of low and high dose, authors supposed that daily food intake might be enough to bring maximal effect.

Our analysis presented several limitations. First, the low number of studies included, combined with the small sample size of studies leaded to less conclusive results, notably for inorganic nitrates, NOS substrates and folates.

Secondly, methodological heterogeneity between the selected studies could interfere with analysis. Techniques of measurement of FMD differed, well reflected by the range of FMD value from 2.3 % to 17.8% with an unexplained value at -0.25 at baseline [START_REF] Bode-Boger | Oral Larginine improves endothelial function in healthy individuals older than 70 years[END_REF]. In one unpublished study (Anisha Pargal 2017 ( 43)), artery measure was unknown. Cuff placement was unknown in 5 studies, in arm in 7 studies and 43 in the forearm. However, metaregressions on baseline FMD were not significant thus the impact of such heterogeneity is probably limited on the results. Another issue was the expression of FMD, some studies expressed in mean difference of dilatation against baseline (in percentage or millimeter), some other expressed overall effect without baseline and few presented mean diameters of the

COMPARAISON OF NUTRITIONNAL STRATEGIES TARGETING THE NITIC OXYDE PATHWAY TO IMPROVE MACROVASCULAR FUNCTION: A SYSTEMATIC REVIEW AND A META ANALYSIS

ABSTRACT: In endothelial cells, production of Nitric Oxide (NO) by NO synthase (NOS) lead to vasodilatation. In many pathologies associated with vascular dysfunction (diabetes, cardiovascular diseases, scleroderma…) this pathway is impacted.

Our main outcome was flow mediated dilatation FMD, used as a surrogate of vascular efficiency and reflecting the endothelial NO release. We studied healthy participants to avoid confounding induced by pathological states. Predefined protocol was registered on PROSPERO (CRD42018090965) and followed PRISMA statement. We searched for double-blind randomized controlled trials (RCTs) in Medline and clinicaltrial.gov up to July 4th, 2018. Evaluated strategies were NOS substrates: L-arginine and L-citrulline, folic acids, inorganic nitrates and polyphenol, supplied by dietary ingredient or nutrient supplement. The secondary outcome was nitrate mediated dilatation (NMD).

55 studies were included, 24 in acute and 38 in chronic supplementation. Acute and chronic efficiency of inorganic nitrates and polyphenols were significant on FMD. Among polyphenols, cocoa displayed the larger effect size. No significant result was found for NOS substrates, folic acids, for all on NMD and on placebo effect. Significant meta regression were on funding, duration of supplementation and smoking. The results were consistent in sensitivity analysis excluding low quality studies.

MOTS CLÉS : méta