Nicolas Lecat

Annie Fourrier-Reglat

PhD Ilaria Montagni

PhD Christophe Tzourio

PhD Antoine Pariente

Helene Verdoux

MD PhD Marie Tournier
email: mtournier@ch-perrens.fr

MD MSc Annie Fourrier-Réglat

PhD Hélène Verdoux

Association between anxiolytic/hypnotic drugs and suicidal thoughts or behaviors in a population-based cohort of students

Keywords: Suicide, students, anxiolytic, hypnotic

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

Introduction

Young adults are vulnerable to suicidal thoughts and behaviors with a short-and long-term impact [START_REF] Goldman-Mellor | Suicide attempt in young people: A signal for long-term healthcare and social needs[END_REF][START_REF] Mortier | The impact of lifetime suicidality on academic performance in college freshmen[END_REF]. According to the World Health Organization (WHO, 2015), suicide is the second cause of death among people 15-29 years old worldwide. Suicide attempts are also particularly frequent in this age group [START_REF] Kessler | Prevalence of and risk factors for lifetime suicide attempts in the National Comorbidity Survey[END_REF].

Thus, a meta-analysis estimated that 3.2% of students presented with a history of lifetime suicide attempt, and 10.6% with suicidal thoughts in the previous 12 months [START_REF] Mortier | The prevalence of suicidal thoughts and behaviours among college students: a meta-analysis[END_REF]. It therefore appears relevant to identify risk and protective factors for suicidal thoughts and behavior in this population such as prescription opioid misuse (Davis et al., 2019). Indeed, many drugs are labeled with warnings from regulatory agencies related to suicide risk. Paradoxically, many of these drugs are psychotropic drugs that are approved to treat psychiatric disorders or symptoms known to increase the occurrence of suicidal thoughts or behavior [START_REF] Nazir | Identification of suicide-related events through network analysis of adverse event reports[END_REF][START_REF] Tournier | A Need for Personalised Suicidology: Pharmacoepidemiology[END_REF]. Some recent studies have shown that benzodiazepines or Z-drugs have frequently been used prior to suicide death [START_REF] Ahlm | Drowning deaths in Sweden with emphasis on the presence of alcohol and drugs -a retrospective study, 1992-2009[END_REF][START_REF] Darke | Toxicology and Characteristics of Deaths Involving Zolpidem in New South Wales, Australia 2001-2010*: ZOLPIDEM TOXICITY[END_REF], thus suggesting that these drugs might trigger suicidal thoughts and/or behavior [START_REF] Brower | Prescription Sleeping Pills, Insomnia, and Suicidality in the National Comorbidity Survey Replication[END_REF][START_REF] Darke | Toxicology and Characteristics of Deaths Involving Zolpidem in New South Wales, Australia 2001-2010*: ZOLPIDEM TOXICITY[END_REF][START_REF] Dodds | Prescribed Benzodiazepines and Suicide Risk: A Review of the Literature[END_REF][START_REF] Saunders | Editorials The role of psychopharmacology in suicide prevention[END_REF][START_REF] Shih | Benzodiazepine therapy in psychiatric outpatients is associated with deliberate selfpoisoning events at emergency departments-a population-based nested case-control study[END_REF]. These studies found a positive and significant association between benzodiazepines and suicidal behaviors. However, they did not demonstrate any causality owing to methodological issues, such as confounding bias as suicidal behavior is a multifactorial process. Particularly, as suicidal behaviors might be linked to the condition treated, confounding by indication bias might occur [START_REF] Costa | Risk factors for suicidal behavior in a university population in Brazil: A retrospective study[END_REF][START_REF] Mccall | Hypnotic Medications and Suicide: Risk, Mechanisms, Mitigation, and the FDA[END_REF][START_REF] Panes | Use and misuse of benzodiazepines in patients with psychiatric disorders[END_REF][START_REF] Zeng | Depressive symptoms, post-traumatic stress symptoms and suicide risk among graduate students: The mediating influence of emotional regulatory self-efficacy[END_REF]. Indeed, no study has shown a prospective association while controlling for depression, insomnia and anxiety symptoms [START_REF] Mccall | Hypnotic Medications and Suicide: Risk, Mechanisms, Mitigation, and the FDA[END_REF]. Furthermore, previous studies did not take into account other risk factors that may be associated both with medication use and suicide, e.g. substance use or temperament traits such as impulsivity, aggression and coping strategies [START_REF] Conner | Metaanalysis of Mood and Substance Use Disorders in Proximal Risk for Suicide Deaths. Suicide Life[END_REF][START_REF] Gvion | Aggression, impulsivity, and suicide behavior: a review of the literature[END_REF].

Mechanisms that might be involved in a potential causal association remain unclear. They might be direct (disinhibition, impairment of judgement through cognitive disturbances, dissociative behavior) or indirect (via depression, rebound insomnia or anxiety) [START_REF] Mccall | Hypnotic Medications and Suicide: Risk, Mechanisms, Mitigation, and the FDA[END_REF]. Some authors suggested that measuring this association might lead to a nocebo effect if patients knew about this potential and rare adverse event [START_REF] Ruan | Commentary: Hypnotic Medications and Suicide: Risk, Mechanisms, Mitigation, and the FDA[END_REF]. It was suggested that drugs targeting some GABA-A receptors might be involved [START_REF] Mccall | Hypnotic Medications and Suicide: Risk, Mechanisms, Mitigation, and the FDA[END_REF]. The roles of repeated withdrawal and of long-term lowering of the brain activation level that might limit the ability to compensate some pre-existing cognitive disturbances remain unknown [START_REF] Pariente | The Benzodiazepine-Dementia Disorders Link: Current State of Knowledge[END_REF]. Moreover, the relationship between zolpidem and suicide was shown cumulative, starting after 80 months of exposure [START_REF] Cho | Temporal association between zolpidem medication and the risk of suicide: A 12-year population-based, retrospective cohort study[END_REF]. Similarly, patients with anxiety disorders had a decreased risk of suicide when they filled benzodiazepines in short-moderate compared with long-term treatment (Boggs, 2019). A dose effect was also shown with zolpidem related to the suicide risk in general population (Sun, 2016). Finally, concurrent use of benzodiazepines, antidepressants, and opioid analgesics with zolpidem increased the risk of suicide compared with the use of zolpidem alone (Sung HG, 2019). It was equally suggested that the use of other substances such as alcohol or MDMA might increase the risk of dissociative or impulsive behavior [START_REF] Darke | Toxicology and Characteristics of Deaths Involving Zolpidem in New South Wales, Australia 2001-2010*: ZOLPIDEM TOXICITY[END_REF].

The use of anxiolytic and hypnotic drugs is not infrequent in young adults. In the USA, 2.6% of people age 18-35 were prescribed benzodiazepine in 2008 with 14.7% of long-term use [START_REF] Olfson | Benzodiazepine Use in the United States[END_REF]. In college students, 1% and 2% of college students received anti-anxiety and sleep medication drugs respectively [START_REF] Eisenberg | Help-Seeking and Access to Mental Health Care in a University Student Population[END_REF]. The College Student Life Survey (CSLS) was conducted during a one-month period, drawing on the total undergraduate population of full-time students attending a large public research university located in the Midwest United States in the winter semesters of the 2003, 2005[START_REF] Eisenberg | Help-Seeking and Access to Mental Health Care in a University Student Population[END_REF], 2009[START_REF] Gvion | Aggression, impulsivity, and suicide behavior: a review of the literature[END_REF]and 2013school years (McCabe et al., 2014)).

Lifetime medical use of sleeping medication increased from 6.4% to 7.4%, and past-year medical use varied between 2.9% in 2003 and 4% in 2009. Lifetime medical use of anxiety medication increased from 6.7 % to 8.8% and past-year medical use from 2.9% to 4.3%. As suicidal thoughts and behaviors are a major public health concern in this population, it is of utmost importance to clarify the role of anxiolytic and hypnotic drugs with respect to suicide risk, taking numerous confounding factors into account.

The study was conducted to assess the association between the use of prescribed anxiolytic or hypnotic drugs and suicidal thoughts or behaviors in a cohort of university students. To minimize confounding by indication, we took numerous risk factors for suicide behaviors into account, in particular psychiatric symptoms or disorders that might require these treatments such as depression, anxiety, impulsivity, aggression, substance use and insomnia. Secondary objectives were to explore the association (i) between the use of anxiolytic or hypnotic drugs at baseline and the occurrence, persistence or remission of suicidal thoughts or behaviors at follow-up, and (ii) between the history of suicidal thoughts or behaviors at baseline and the occurrence of anxiolytic or hypnotic drug use at follow-up.

Methods

Study design and study population

Cross-sectional assessments were conducted in a national prospective population-based cohort study with public funding, the ongoing Internet-based Students Health Research Enterprise (i-Share) project [START_REF] Montagni | Screen time exposure and reporting of headaches in young adults: A cross-sectional study[END_REF]. This project assesses students' health in French-speaking universities and higher education institutions by self-administered online questionnaires (Figure S1).

Students register voluntary for the study and complete a mandatory baseline questionnaire online.

An optional mental health survey is sent to them three months after completing the baseline questionnaire in order to investigate psychiatric disorders, personality and temperament traits using psychometric scales (see section "Other collected variables"). A follow-up questionnaire is then sent to them one year after inclusion to assess health care use, suicidal thoughts and behaviors among other items. Inclusion criteria for the i-Share cohort are the following: i) officially registered at a university or higher education institute, ii) at least 18 years old, iii) able to read and understand French. All participants provided informed consent. The study population included all participants who fully completed the baseline questionnaire between April 2013 and March 2017. Within the cohort, three populations were studied independently: all included students (i.e. students having completed the baseline questionnaire), those also having completed the optional mental health questionnaire and those having completed both baseline and follow-up questionnaires.

Exposure to anxiolytic/hypnotic drug, suicidal thoughts and behaviors

The main variables were exposure to anxiolytic/hypnotic drugs and suicidal thoughts and behaviors; points of measurement over the study period are shown in Figure 1. They were explored through the baseline and follow-up questionnaires. Drug use was assessed within the previous three months at each contact. Anxiolytic drugs were defined as medications "against anxiety or stress (during the day)" and hypnotics as medications "to sleep": "Within the past 3 months, have you ever taken medications for anxiety, anguish or stress (in the day time) / to sleep? Was this drug obtained on medical prescription?". Drug classes and names of products were unknown. Drugs were considered only when they were reported as prescribed to the participant by a physician. Thus, drugs obtained over the counter were not considered. The variable of interest was the use of at least one prescribed anxiolytic or hypnotic drug over the 3-month period.

The baseline questionnaire investigated the presence of suicidal thoughts over the previous 12 months ("Over the 12 preceding months, have you ever thought about killing yourself (suicidal thoughts)?") as well as lifetime suicide attempts ("Over your lifetime, did you ever attempt suicide?"). Time windows were different since suicidal thoughts are more common and considered as less severe than suicide attempts. Suicide attempts are the main risk factor for suicide, even several years after [START_REF] Bostwick | Suicide Attempt as a Risk Factor for Completed Suicide: Even More Lethal Than We Knew[END_REF][START_REF] Suominen | Completed suicide after a suicide attempt: a 37-year follow-up study[END_REF]. The variable "suicidal thoughts and/or behaviors (STB)" was defined as the presence of at least one of the two factors. To select students at high and current suicide risk, a variable "suicidal thoughts and behaviors" was considered for sensitivity analyses and corresponded to students who had both suicide thoughts over the previous 12 months and at least a lifetime suicide attempt. The follow-up questionnaire investigated the presence of suicidal thoughts and attempts over the previous 12 months. Incident STB were identified in the sub-population having completed the follow-up questionnaire and were defined as the presence of suicidal thoughts and/or behaviors at follow-up (thoughts and/or attempt over the previous year) and none at baseline. In the same sub-population, persistent STB corresponded to the presence of STB at baseline and the presence of suicidal thoughts and/or suicide attempt over the previous year at follow-up, while remission corresponded to the presence of STB at baseline and its absence over the previous year at follow-up.

Other collected variables

Covariates were collected through the baseline and the mental health questionnaires. We selected a priori risk factors for STB that could also be associated with use of anxiolytics or hypnotics to limit confounding. They related to participants' characteristics: i) sociodemographic characteristics: age, sex, year of study (categorized in four groups: 1 st , 2 nd , 3 rd or higher year of university), scholarship, siblings, accommodation (with his/her parent(s), university residence, apartment), living alone, being an orphan, divorced parents, paid employment; ii) dissatisfaction with living conditions: regarding recreational opportunities, relationships with parents, social life and financial resources; iii) use of health care over the previous year: at least one consultation with a general practitioner (GP), at least one consultation with a psychiatrist, psychologist or psychotherapist, avoiding an appointment with the GP or not buying prescribed medication although it was necessary/recommended; iv) health status: disability, at least one physician-diagnosed somatic disease, at least one physician-diagnosed psychiatric disease, and exposure to a traumatic life event causing intense fear; v) family history: parental history of depression, anxiety disorders or alcoholism; vi) health behaviors: unhealthy BMI (≤18kg/m 2 , 18-30, ≥30kg/m 2 ), regular physical exercise (WHO, n.d.), practicing a team sport; vii) sleep disturbances: global sleep quality (poor/not poor), difficulty in falling asleep/maintaining sleep (more/less than once a week), feeling extremely sleepy during the day (more/less than 3 to 5 times per week), usual lack of sleep with at least one hour less than necessary (more than a few times a week/once a week or less); and viii) substance use: smoking status, alcohol use (more than 4 times a week/3 times a week or less), cannabis use (more/less than once in the previous year), at least one lifetime use of another substance, or at least one use of doping products during competitions or exam periods. The use of other psychotropic medication drugs was not available in the database.

The mental health questionnaire consisted of nine psychometric scales: a perceived stress assessment adapted to the student population (17 items based on four main factors: academic stress, dysfunctions at university, feelings of loneliness, and problems with close relations) [START_REF] Boujut | A construction and validation of a freshman stress questionnaire: an exploratory study[END_REF]; a scale assessing coping strategies in 16 items divided in three parts: search for social support, emotional coping, festive and addictive coping [START_REF] Boujut | Coping among Students: Development and Validation of an Exploratory Measure[END_REF]; the Spielberger trait anxiety inventory (STAI-YA): feelings of apprehension, tension, nervousness and anxiety [START_REF] Spielberger | Psychometric properties of the STAI: a reply to Ramanaiah, Franzen, and Schill[END_REF]; the adolescent version of the Barratt Impulsivity Scale (BIS-11-A) based on a three-dimensional structure: motor (behavior), attentional (cognitive) and lack of planning [START_REF] Baylé | Factor analysis of french translation of the Barratt impulsivity scale (BIS-10)[END_REF]; the Life History of Aggression (LHA) including nine items considering aggressive behavior in adolescence and adulthood [START_REF] Coccaro | Assessment of life history of aggression: development and psychometric characteristics[END_REF]; the Patient Health Questionnaire (PHQ-9) based on DSM IV diagnostic criteria [START_REF] Arthurs | Are Scores on English and French Versions of the PHQ-9 Comparable? An Assessment of Differential Item Functioning[END_REF][START_REF] Kroenke | The PHQ-9: A New Depression Diagnostic and Severity Measure[END_REF] to assess a current major depressive episode of at least moderate intensity (score >10); the Mood Disorder Questionnaire (MDQ) to screen bipolar disorders (at least 7 lifetime manic or hypomanic symptoms, several co-occurring symptoms, and moderate or serious associated functional impairment) [START_REF] Weber Rouget | Screening for bipolar disorders using a French version of the Mood Disorder Questionnaire (MDQ)[END_REF]; the Rosenberg self-esteem scale, which includes 10 items on a four-point Likert scale [START_REF] Alessandri | On the factor structure of the Rosenberg (1965) General Self-Esteem Scale[END_REF]; and a short version of Cloninger's Temperament and Character Inventory (TCI) with 56 items that allow the creation of 6 scores, including temperament traits (novelty-seeking, harm avoidance, dependence on reward and persistence), and character traits (determination and cooperation) [START_REF] Pelissolo | The Temperament and Character Inventory-Revised (TCI-R): psychometric characteristics of the French version[END_REF].

Concerning MDQ and PHQ-9 scores, validated thresholds made it possible to obtain binary variables (presence/absence). For the other scales, quantitative scores were transformed into binary variables (high/low) using the median obtained in the study population as threshold, since thresholds validated in different populations might not be appropriate in students.

Statistical method

All statistical analyses were performed by using SAS (version 9.4; SAS Institute Inc, Cary, NC, USA) statistical software. First, we carried out descriptive analyses of the study population.

Participants who had also completed the mental health questionnaire were compared with those who did not using univariate tests, the Pearson Chi2 test for qualitative variables and the Student test for quantitative variables. Completers of the follow-up questionnaire were compared with noncompleters. Second, covariates were summarised in disease risk scores (DRS) in order to control for a large number of confounding variables [START_REF] Tadrous | Disease risk score as a confounder summary method: systematic review and recommendations: DRS AS A CONFOUNDER SUMMARY METHOD[END_REF]. Then, several multivariate logistic regression models were run to assess the association between prescribed anxiolytic/hypnotic drugs and STB.

The first model assessed the association between STB and the use of prescribed anxiolytic/hypnotic drugs at baseline in the whole population, adjusting for a DRS including all covariates explored through the baseline questionnaire. Three sensitivity analyses were performed for this model: i) for the first one, "suicidal thoughts and/or behaviors", i.e. STB, was replaced by "suicidal thoughts and behaviors"; ii) the second one was conducted in participants having completed the optional mental health questionnaires to add these covariates to the DRS for this model; and iii) for the third one, all covariates were individually included in the model (TableS1).

The three other models were run for participants who had completed the follow-up questionnaire. The first one assessed the association between incident STB (absence at baseline and presence at follow-up) and the use of prescribed anxiolytics/hypnotics at baseline, adjusting for a DRS including all covariates explored through the baseline questionnaire, in students without STB at baseline. The second one assessed the association between persistent STB (presence at baseline and at follow-up) and the use of prescribed anxiolytics/hypnotics at baseline, adjusting for a DRS including all covariates explored through the baseline questionnaire, in students with STB at baseline.

The last one investigated the association between the use of prescribed anxiolytic/hypnotic drugs at follow-up and STB at baseline, adjusting for a DRS including all covariates explored through the baseline questionnaire. For these models, sensitivity analyses were performed individually including covariates in the models (Table 5).

All covariates were defined a priori according to their clinical relevance. Variables with more than 20% missing data were eliminated from all models. For sensivity analyses individually including all covariates, all interactions were tested and the suitability of the model was verified with the Hosmer-Lemeshow test, and its discriminating power was calculated by computing the area under the ROC curve.

Results

A total of 12,112 students completed the i-Share baseline questionnaire. Among them, 3,585 students fully completed the mental health questionnaire and were then identified as the "mental health subgroup". Finally, 2,919 students completed the follow-up questionnaire and were considered as the "follow-up subgroup". Flow chart is shown in Figure 1.

Description of study population

Among the 12,112 students enrolled in the i-Share cohort, 97.5% students answered questions on suicide thoughts and attempts (n=11,588). STB were identified in 2,916 students (25.2%), nearly 22% reporting suicidal thoughts in the past 12 months, and 6.2% a lifetime suicide attempt (Table 1). These students are described in Table 2 according to the presence or not of STB. In both groups, most participants were women, and more than one-third were in the first year. Both groups were similar in terms of age (mean 20.4 years; SD 2.6). Students with or without STB at baseline had different characteristics regarding nearly all sociodemographics, health care use and health status, apart from age, having siblings and a paid job. In particular, the use of prescribed anxiolytic and/or hypnotic was nearly three times higher among students with STB than among students without STB (Table 2).

Description of sub-population who completed the mental health questionnaire

The sub-sample who completed the mental health questionnaire was compared to the students who did not (data not shown). Completers were slightly different from the others. The main differences concerned scholarship (62 % in whole group vs 67 % in mental health subgroup, p<0.0001) and smoking (34 % vs 26 %, p<0.0001). Almost all mental health scores were very different in students with or without STB at baseline, with poorer mental health in students with STB than in those without (Table 3). However, seeking social support as a coping strategy and planning difficulties were similar across the groups, as well as some temperament traits such as noveltyseeking, harm avoidance and reward dependence.

Cross-sectional association between use of anxiolytic/hypnotic drugs and STB

STB at baseline were associated with the use of prescribed anxiolytic/hypnotic medications at baseline, after adjustment for all covariates (Table 4). This association persisted in sensitivity analyses after adjustment for mental health characteristics in the subgroup of students who completed the mental health questionnaire. The co-existence of suicidal thoughts and behaviors was also associated with the use of anxiolytic/hypnotic drugs. Similar results were found when covariates were individually included in the models sensitivity analyses (Table 5).

Longitudinal associations between use of anxiolytic/hypnotic drugs and STB

At the follow-up, incident STB were 7.1% and almost 47% of students with STB at baseline still had them. The sub-sample who completed the follow-up questionnaire was compared to students who did not (data not shown). Completers were slightly different from the others. The main differences concerned scholarship (60 % among non-completers vs 78 % among follow-up subgroup, p<0.0001) and smoking (35 % vs 26 %, p<0.0001).

No association was found between the prescription of anxiolytic/hypnotic medication at baseline and incident STB one year later in students without STB at inclusion or the persistence of STB at follow-up (Table 4). On the contrary, there was a significant association between STB at baseline and a new use of prescribed anxiolytic/hypnotic drugs at follow-up (aOR 1.80; 95%CI 1.20-2.71). This model showed a 80% increase in new use of prescribed anxiolytic/hypnotic at follow-up after self-reported STB at baseline. The same result was found when covariates were individually included in the model in sensitivity analysis (aOR 1.9; 95%CI 1.1-3.3).

Discussion

Main findings

This study conducted in a large student population is the first one to show an association between the use of prescribed anxiolytic/hypnotic medication and STB, even when considering psychiatric disorders and symptoms including depression, anxiety, perceived stress, sleeping disorders, impulsivity and substance use. Another striking finding is the high number of students found to have STB.

Indeed, more than one out of five students reported suicidal thoughts over the year preceding the baseline questionnaire. This rate was higher than in a recent meta-analysis assessing suicidal thoughts among college students (10.6%, 95%CI 9.10;12.25) [START_REF] Mortier | The prevalence of suicidal thoughts and behaviours among college students: a meta-analysis[END_REF], but similar to the frequency described among US undergraduate students (24%) [START_REF] Liu | The prevalence and predictors of mental health diagnoses and suicide among U.S. college students: Implications for addressing disparities in service use[END_REF] or in medical students over the same duration (24.2%, 95%CI 13.0;40.5) [START_REF] Rotenstein | Prevalence of Depression, Depressive Symptoms, and Suicidal Ideation Among Medical Students: A Systematic Review and Meta-Analysis[END_REF]. The frequency of lifetime suicide attempts was also nearly two-fold higher in the present study than in the findings of the meta-analysis (1.2%, 95%CI 0.83;1.59) [START_REF] Mortier | The prevalence of suicidal thoughts and behaviours among college students: a meta-analysis[END_REF] but lower than in a study using the same design (9%) [START_REF] Liu | The prevalence and predictors of mental health diagnoses and suicide among U.S. college students: Implications for addressing disparities in service use[END_REF]. As inclusion in the i-Share cohort is voluntary and its objective is to assess students' health conditions, students concerned about health or having health problems, especially mental health disorders, might be over-represented in the sample. However, we cannot exclude an especially high frequency of suicide behaviors in French students compared with other countries, as already reported for deliberate self-harm [START_REF] Brunner | Life-time prevalence and psychosocial correlates of adolescent direct self-injurious behavior: A comparative study of findings in 11 European countries[END_REF]. STB at baseline were associated with the use of a prescribed anxiolytic or hypnotic drug at inclusion when taking all other variables into account, including psychiatric disorders, anxiety, sleep disturbances, impulsivity and substance use. This minimized confounding and particularly confounding by indication. The association persisted when considering the co-existence of suicidal thoughts and behaviors. When students presented a history of suicide attempt and recent suicidal thoughts, they might be offered care more easily or they might be more likely to seek professional support.

STB at baseline were also associated with a new prescription of anxiolytic/hypnotic drugs at follow-up. Students with mental distress, and particularly suicidal thoughts or attempts, were likely to seek mental health care and, among other treatments, to receive a prescription for anxiolytic/hypnotic drugs. This request for health care might be deferred and be formulated later than suicidal thoughts, because students might lack information about the availability of health care services [START_REF] Montagni | Avoidance and Delay of Medical Care in the Young: An Interdisciplinary Mixed-methods Study[END_REF], or they might be worried about or suspicious of psychiatry and psychotropic drugs. They might wait until their distress is overwhelming before consulting.

Moreover, only a few students with STB sought health care. Conversely, we did not find any association between the use of prescribed anxiolytic/hypnotic medication within the three months preceding the baseline questionnaire and the occurrence of STB or the persistence or remission at follow-up of STB already identified at the time of inclusion [START_REF] Ribeiro | Selfinjurious thoughts and behaviors as risk factors for future suicide ideation, attempts, and death: a meta-analysis of longitudinal studies[END_REF]. This suggests that the use of these drugs did not attenuate the students' STB.

Following the increase in suicide rates among young adults, the association between suicide and prescription drug use and misuse was assessed by few studies. Non-medical use of prescription drugs increased over years in college students except from painkillers [START_REF] Mccabe | Trends in medical use, diversion, and nonmedical use of prescription medications among college students from 2003 to 2013: Connecting the dots[END_REF]. A recent study carried on in college students found an association between suicidal ideation, suicide planning or suicide attempts with prescription opioid misuse (any use not prescribed) [START_REF] Davis | Association between prescription opioid misuse and dimensions of suicidality among college students[END_REF].

Another study considered misuse of several drug classes and showed a unique association between suicidal ideation and misuse of stimulants [START_REF] Zullig | The association between non-medical prescription drug use, depressive symptoms, and suicidality among college students[END_REF]. In particular, misuse of benzodiazepines was not associated with suicidal ideation or suicide attempts. However, none of these studies investigated the impact of medical use of anxiolytics or hypnotics on STB in students.

Limitations and strengths

These longitudinal analyses were performed in an exploratory way and several methodological limitations might have hampered the identification of such an association. A depletion of susceptible bias might be suspected, since considering only the occurrence of incident STB at follow-up excluded all students who presented with suicidal thoughts in the year prior to inclusion and who used anxiolytic/hypnotic drugs in the preceding three months; in other words, all students likely to present suicidal thoughts and/or behaviors when exposed to these drugs. Furthermore, a selection bias might be suspected, as many students were lost to follow-up.

However, only slight differences were identified between completers and non-completers. Some other limitations should be considered. First, the abovementioned selection bias (registration in the cohort on a voluntary basis) might have modified the association only if it was differential. Second, students less likely to participate might have been students with STB [START_REF] Mortier | Suicidal thoughts and behaviors among college students and same-aged peers: results from the World Health Organization World Mental Health Surveys[END_REF] and nonusers of prescribed medications, which would tend to reduce the strength of the association that we identified. Third, there may have been a competitive risk because students who died by suicide were not identified. In fact, there was a 97% risk that a maximum of two students might have died from suicide during the follow-up. Fourth, the vagueness of the questions concerning drug use may have created an information bias. However, to minimize this risk, we considered only prescribed medications in order to exclude over-the-counter drugs such as herbal medicine and to select highpotency drugs. Similarly, STB were not assessed with a validated scale. Moreover, misuse of anxiolytics/hypnotics, defined as a use non-compliant with guidelines, was not considered in the study. A misuse similar to the one of opioid painkillers could happen among our population study and might have led to overestimate the association between STB and drug use. This bias was minimized as only drug use from medical prescription was selected, excluding the one borrowed from the entourage or used over the counter. Finally, residual confounding by indication bias might have led us to conclude erroneously about an association between drug use and suicide risk, since these drugs and suicide risk might be independently associated with the condition treated. Although most indications of these drugs were adjusted for, as well as other confounding factors, further studies are warranted to investigate clinical events occurring when such treatments are initiated and during the following weeks [START_REF] Millner | Describing and Measuring the Pathway to Suicide Attempts: A Preliminary Study[END_REF].

The main strengths of the study are its large number of participants and the number of explored variables. Many risk factors were assessed with validated tools including psychiatric disorders and symptoms, substance abuse and chronic somatic diseases. Many suicide protective factors such as social and family support and coping strategies were also investigated. In fact, the study was conducted in a large cohort who completed many relevant items. The use of a composite criterion (thoughts and/or behaviors) made it possible to consider those who had made an attempt without suicidal thoughts, which can be associated with the use of medication in youth [START_REF] Copelan | Impact of nonideation states on youth suicide attempts[END_REF]. Finally, the online self-administered assessment could be a strength by circumventing disclosure barriers such as shame.

In conclusion, this large community-based cohort study found a cross-sectional association between the use of anxiolytics/hypnotics and STB, with an approximately 50% increase in the risk of the latter. This association was independent from many risk factors of suicide and from most psychiatric disorders that require such treatments. Even if residual confounding cannot be excluded, these findings should alert prescribers to this potential risk and at least to the lack of demonstrated effectiveness of these drugs on suicidal thoughts or behaviors and encourage them to weigh up the expected benefit and risks when prescribing anxiolytics or hypnotics, even to young patients. Therapeutic alternatives might be considered such as psychotherapy and other psychotropic drugs when relevant.

Table 1 .

 1 Suicidal thoughts and behaviors at baseline and at follow-up

	Number of students with
	available information	N	(%)

At time of follow-up in students with suicide risk at baseline (n=718)

	Persistent suicidal thoughts and/or behaviors c	685	321	46.9

At time of follow-up in students with no suicide risk at baseline (n=2,106)

  suicidal thoughts in preceding year and/or lifetime suicide attempt; b suicidal thoughts in preceding year and lifetime suicide attempt; c Suicidal thoughts and/or behaviors at baseline and suicidal thoughts and/or attempt in preceding year at follow-up; d No suicidal thoughts nor behaviors at baseline and suicidal thoughts and/or attempt in preceding year at follow-up.

	Suicidal thoughts in previous 12 months	2,029	139	6.8
	Suicide attempt in previous 12 months	2,037	6	0.3
	Incident suicidal thoughts and/or behaviors d	2,029	145	7.1

a

Table 2 .

 2 Description of the whole study population according to presence of suicidal thoughts and/or

	behaviors at baseline (n=11,588)
			Students without	Students with STB	Chi-2
			STB (n=8,672)	(n=2,916)	T-test
		n a	n (%)	n (%)	p
	Prescribed anxiolytics/hypnotics in
		11,588	643 (7.4)	554 (19.0) <0.0001
	preceding 3 months
	Sociodemographic characteristics
	Age [mean (SD)]	11,588	20.4 (2.6)	20.4 (2.7)	0.67
	Sex (woman)	11,588	6,412 (73.9)	2,254 (77.3) 0.0003
	Year of study	11,588			<0.0001
	1		3,321 (38.3)	1,289 (44.2)
	2		1,691 (19.5)	556 (19.1)
	3		1,324 (15.3)	394 (13.5)
	4 or more		2,336 (26.9)	677 (23.2)
	Scholarship	11,316	5,361 (63.2)	1,898 (67.0) 0.0003
	Siblings	11,459	7,838 (91.4)	2,611 (90.5)	0.1
	Accommodation	11,588			<0.0001
	With his/her parent(s)		2,770 (31.9)	868 (29.8)
	University residence		943 (10.9)	428 (14.7)
	Apartment		4,655 (53.7)	1,503 (51.5)
	Orphan (at least one parent)	11,588	413 (4.8)	179 (6.1)	0.004
	Divorced parents	11,235	2,502 (29.7)	1,095 (39.0) <0.0001

Table 2 .

 2 Description of the whole study population according to presence of suicidal thoughts and/or

	behaviors at baseline (n=11,588) (continued)
	Paid employment	11,588	2,951 (34.0)	1,010 (34.6)	0.6
	Dissatisfaction with living conditions
	Relationship with parents	11,588	634 (7.3)	567 (19.4) <0.0001
	Financial resources	11,588	1,614 (18.6)	837 (28.7) <0.0001
	Recreational opportunities	11,588	1,885 (21.7)	954 (32.7) <0.0001
	Social life	11,588	1,215 (14.0)	787 (27.0) <0.0001
	Use of health care over previous year
	Consultation with GP	11,587	7,436 (85.7)	250 (87.1)	0.07
	Consultation with mental health
		11,467	741 (8.6)	770 (26.8) <0.0001
	professional
	Avoiding GP consultation	11,588	2,804 (32.3)	1,445 (49.6) <0.0001
	Not buying prescribed medications	11,588	1,726 (19.9)	926 (31.8) <0.0001
	Health status
	Somatic illness b	11,588	3,030 (34.9)	1,257 (43.1) <0.0001
	Psychiatric illness c	11,588	1,417 (16.3)	1,250 (42.9) <0.0001
	Parental history of depression
		10,243	3,321 (42.9)	1,614 (64.7) <0.0001
	/anxiety/alcohol use disorder
	Disability	11,588	230 (2.7)	128 (4.4) <0.0001
	Traumatic event that caused
		11,481	2,646 (30.8)	1,315 (45.6) <0.0001
	intense fear

Table 2 .

 2 Description of the whole study population according to presence of suicidal thoughts and/or behaviors at baseline (n=11,588) (continued) at least one lifetime use of another substance among ecstasy, MDMA, amphetamines, nitrous oxide, inhalant, cocaine, magic mushrooms (or other hallucinogenic plants), crack, free-base, heroin, LSD, or ketamine STB: suicidal thoughts and/or behaviors; GP: general practitioner Table3. Description of mental health characteristics according to presence of suicidal thoughts and/or behaviors at baseline(n=3,585)

	Health behavior
	Physical regular activity	11,566	8,135 (94.0)	2,667 (91.7) <0.0001
	Practicing a team sport	11,588	1,235 (14.2)	302 (10.4) <0.0001
	BMI	10,962			<0.0001
	≤ 18 kg/m2		706 (8.6)	277 (10.1)
	18 -30 kg/m2		7,323 (89.7)	2,344 (85.6)
	≥ 30 kg/m2		194 (2.36)	118 (4.3)
	Sleep disturbances
	Poor quality of sleep	11,588	1,556 (17.9)	949 (32.5) <0.0001
	Difficulty falling asleep/maintaining
		11,585	3,522 (40.6)	1,703 (58.4) <0.0001
	sleep
	Daytime sleepiness	11,586	3,586 (41.4)	1,712 (58.7) <0.0001
	Usual lack of sleep	11,588	4,227 (48.7)	1,764 (60.5) <0.0001
	Substance use
	Doping products	11,588	1,874 (21.6)	776 (26.6) <0.0001
	Tobacco	11,588	2,468 (28.5)	1,114 (38.2) <0.0001
	Alcohol	11,588	351 (4.1)	150 (5.1)	0.01
	Cannabis	9,187	2,143 (31.2)	969 (41.9) <0.0001
	Other drugs d	11,570	3,016 (34.8)	1,085 (37.3)	0.02
	a Number of students with available information; b including migraine, chronic inflammatory bowels
	disease, multiple sclerosis, type 1 diabetes, asthma, juvenile arthritis; c including depression,
	attention deficit/hyperactivity disorder, obsessive compulsive disorder, anxiety disorders or
	anorexia;

d

Table 4 .

 4 Association between use of anxiolytic or hypnotic drugs at baseline and suicidal thoughts and/or behaviors at baseline or at follow-up (multivariate models) Number of students with available information; b Number of students with suicidal thoughts and/or behaviors ; c Odds ratio adjusted on all covariates: sociodemographic characteristics, dissatisfaction with living conditions, use of health care over previous year, health status, health behaviors, sleep disturbances and substances use; c Odds ratio adjusted on all these covariates and on perceived stress, coping strategies, trait-anxiety, impulsivity, aggression, current major depressive episode, bipolar disorder, self-esteem, temperament and character traits. STB: suicidal thoughts and/or behaviors

		Use of anxiolytic or hypnotic drugs
	N a	n b
			at baseline
		aOR c	95%CI

a

Acknowledgements

The authors thank Ray Cooke for copyediting the manuscript. The authors are indebted to the participants of the i-Share project for their commitment and cooperation and to the entire i-Share team for their expert contribution and assistance.

Declaration of interests:

Authors have no conflict of interest to disclose.

Funding information

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Nicolas Lecat was a recipient of an award from the GEPS (Groupement d'études et de prevention du suicide for Group for Suicide Studies and Prevention) in France to fund his master's degree.

Ethics approval

The i-Share project from which this study was derived was approved by the Commission Nationale de l'Informatique et des Libertés (CNIL) [DR-2013-019].


C Courbon

I Arrieta

F Cabanettes

J Rech

P.-J Arrazola

The Contribution of Microstructure and Friction in Broaching Ferrite-Pearlite Steels

Keywords: Cutting, Micro structure, Friction, Chip

Ferrite-Pearlite (FP) steels are used in many automotive components. However, their machinability in low cutting speed processes appears to be highly dependent on their metallurgical state. An experimental approach combining broaching and tribological tests under machining-like conditions was developed to determine the key FP features driving machining performance. Fundamental tests were performed on fifteen variants so as to cover a wide range of microstructural properties under dry and lubricated conditions using both uncoated and TiN coated High Speed Steel (HSS) tools. The correlation between the microstructure, tribology and outputs such as machining forces and chip thickness ratio is presented.

Introduction

Carbon steels are used in many automotive components especially transmission shafts, gear wheels, pinions or wheel hubs, where the core of the workpiece is primarily composed of a Ferrite-Pearlite (FP) microstructure.

FP steels are considered as conventional materials but they are not the easiest materials to machine. The low deformability and greater hardness of pearlite has been reported to cause significant abrasive wear and high resultant forces [START_REF] Doane | Softening high hardenability steels for machining and cold forming[END_REF] [START_REF] Ebrahimi | Evaluation of machinability in turning of microalloyed and quenched-tempered steels: Tool wear, statistical analysis, chip morphology[END_REF]. On the other hand, pearlite promotes the formation of favourable chips and limited burrs, improved surface quality [START_REF] Ozcatalbas | The effects of heat treatment on the machinability of mild steels[END_REF], to reduce adhesion tendency and the occurrence of built-up edges [START_REF] Akasawa | Effect of microstructure and hardness on the machinability of medium-carbon chrome-molybdenum steel[END_REF].

Broaching is widely used in industry, however there is little published research on this process when compared to turning, milling or drilling. The few studies found in the literature have been conducted to identify the process parameters affecting cutting forces [START_REF] Mo | An example of selection of the cutting conditions in broaching of heat-resistant alloys based on cutting forces, surface roughness and tool wear[END_REF], surface integrity [START_REF] Kishawy | An energy based analysis of broaching operation: Cutting forces and resultant surface integrity[END_REF], surface roughness [START_REF] Arrieta | Influence of the ferritic-pearlitic steel microstructure on surface roughness in broaching of automotive steels[END_REF][START_REF] Fabre | Optimization of surface roughness in broaching[END_REF], the influence of different coatings and lubrication [START_REF] Schulze | High Speed Broaching of Case Hardening Steel SAE 5120[END_REF] and the development of innovative monitoring techniques [START_REF] Axinte | Broaching of Ti-6-4 -Detection of Workpiece Surface Anomalies on Dovetail Slots through Process Monitoring[END_REF]. Moreover, very little work exists on broaching [START_REF] Strauss | Local Residual Stress Distribution at the Tooth Root Surface of a Broached Steel Component[END_REF] or even micro cutting [START_REF] Strauss | Local Residual Stress Distribution at the Tooth Root Surface of a Broached Steel Component[END_REF] of FP steels. To date, no study has been found which specifically investigates the effect of FP microstructural parameters, i.e. ferrite content, grain size and hardness, on machining forces or chip formation in the broaching process.

In addition, findings from [START_REF] Akasawa | Effect of microstructure and hardness on the machinability of medium-carbon chrome-molybdenum steel[END_REF] and [START_REF] Schulze | High Speed Broaching of Case Hardening Steel SAE 5120[END_REF] suggest that tribological phenomena could play a major role in governing the cutting behaviour. Whereas many attempts have been made over the years [START_REF] Melkote | Advances in material and friction data for modelling of metal machining[END_REF] to improve the understanding of friction in cutting, whether by using the cutting process itself [START_REF] Childs | On the Stress Distribution Between the Chip and Tool During Metal Turning[END_REF] [START_REF] Arrazola | A new approach for the friction identification during machining through the use of finite element modeling[END_REF] or laboratory tests [START_REF] Olsson | Simulation of cutting tool wear by a modified pin-on-disc test[END_REF] [START_REF] Rech | Characterisation of friction and heat partition coefficients at the tool-work material interface in cutting[END_REF], no study has clearly reported the exact correlation between friction and the FP microstructure. This work thus aims to identify (i) the exact contribution of FP microstructure on machining outputs, (ii) the influence of FP microstructural parameters on friction and (iii) the correlation between friction and machining outputs in a broaching framework. To this end, an experimental approach was developed combining one tooth fundamental broaching tests with tribological tests under machining-like conditions.

Experimental approach

Investigated materials

Five steel grades, 16MnCr5, 27MnCr5, C45, C60 and C70 with increasing carbon content were selected to generate ferrite ratios from 6 to almost 70%. C70 was also investigated as a fully pearlitic steel for reference purposes. Homogeneous FP microstructures (Fig. 1a) were obtained by a standard isothermal annealing cycle. Processing and heat treatments (Fig. 1b) were applied on each grade to extend the range of microstructural characteristics (Fig. 1c). A specific heat treatment was designed to increase the material grain size (Fig. 1d) whereas globular annealing was developed to achieve an overall softening and increase the ferrite ratio. Cementite lamellas were transformed into spherical particles, uniformly distributed in the predominant ferrite matrix (Fig. 1e). In total, 15 different materials were manufactured and analysed to properly define the ferrite ratio (F%) and macroscopic Brinell hardness. The ferrite ratio was measured using statistical image analysis based on 30 to 40 images taken at a 200x magnification in 10 random locations within the sample. For all the variants except one, a relatively constant grain size was achieved ranging reasonably between 10 and 20µm, limiting its potential effect.

Fundamental broaching test

A dedicated experimental set-up, developed in previous studies focusing on surface roughness [START_REF] Arrieta | Influence of the ferritic-pearlitic steel microstructure on surface roughness in broaching of automotive steels[END_REF][START_REF] Fabre | Optimization of surface roughness in broaching[END_REF], was used to perform fundamental tests up to 60m/min. To alleviate any lack of stiffness, the experimental campaign was conducted with a single tooth broaching tool. Steel specimens were manufactured with a cutting width b = 3mm and fitted in the spindle via s specific holder while a HSS M35 broaching tool was fixed to the machine table. A dynamometer was used to record the main cutting force Fc and normal force Fn.

The broaching tool was designed with a rake angle γ of 17°, a clearance angle α = 1°, a cutting edge radius rβ = 5µm and an edge inclination angle λS = 0°, resulting in orthogonal conditions.

Experiments were run at cutting speeds vc = 2, 20 and 60m/min, with an uncut chip thickness h = 10 and 40µm, under both dry and straight oil lubricated conditions. The results show the average and standard deviation of 5 consecutive runs.

Tribological tests

The tribological investigation was based on an open tribometer mounted on a CNC lathe [START_REF] Rech | Characterisation of friction and heat partition coefficients at the tool-work material interface in cutting[END_REF]. The tool-material interaction was tested by scratching a pin with a spherical geometry, made of HSS M35 (uncoated or TiN coated), against a cylindrical bar of each studied variant. After each friction test, a cutting tool refreshed the scratched surface to remove the affected layer of work material, and a belt finishing operation was then applied.

The sliding velocity Vsl, defined by the rotation speed of the spindle, ranged from 2 to 60m/min, and the normal load was set to 1000N via a hydraulic jack, leading to a contact pressure of around 1.5GPa. Friction pins were ground and polished to achieve a roughness Ra of around 0.3µm.

The apparent friction coefficient was computed as the ratio of the tangential force to the normal load measured by a piezoelectric dynamometer. The results set out the average and standard deviation of three repetitions performed for each sliding condition, each with a new pin.

Interaction under uncoated dry conditions

Broaching tools are usually fully coated and then ground on the rake face in order to accurately control the cutting edge radius. This leads to a specific configuration with a coated flank face and uncoated rake face. To simplify the cutting configuration and dissociate the effect of the microstructure, uncoated tools were first selected under dry conditions. The results of this campaign are displayed versus F% (Fig. 2a,b) and macroscopic hardness in terms of normal force only (Fig. 2c). Considering the large amount of data, second order polynomial equations were fitted to each of the datasets in order to analyse the main trends as presented in Figure 2.

The following quantitative comparisons are given based on this surface fit with a range of F% from 5 to 70% and hardness from 140 to 260HB. Figure 2 summarises the effects of both FP microstructural parameters and cutting conditions.

It first shows that at both h and low cutting speed, increasing F% (Fig. 2a) led to an increase in the main cutting force Fc. At h = 40µm, increasing F% generated an increase of 40% in Fc. This effect was reduced significantly when increasing the cutting speed up to 60m/min. Indeed, a 10% variation of Fc was observed with the same increase in F%. Materials with a larger ferrite content F% = 70% were found to be more sensitive to the cutting speed with a decrease of nearly 20% in Fc when changing from 2 to 60m/min, as opposed to only 5% with a material with a lower ferrite ratio F% = 5%.

When h was reduced from 40 to 10µm, similar amplitudes were reported at 2m/min. An increase of 36% in Fc was observed when increasing F%. Tests conducted at 60m/min exhibited larger variations (+28%) showing the limited effect of the cutting speed with a small uncut chip thickness.

As regards the normal force Fn, a similar trend to Fc was noted with h = 40µm characterised by significant increase of 70% when increasing F% at vc = 2m/min and 36% at vc = 60m/min. This behaviour was seen again to be less dependent on the cutting speed with h = 10µm. In the case of the latter, Fn still increased by 50 to 60% when increasing F% (Fig. 2b), however the cutting speed no longer appeared to play a major role. Fn was indeed reduced by 35% at both 2 and 60m/min when decreasing F% from 70 to 5%. Finally, if machining forces generally increased when increasing F%, Fn appeared to be more affected compared to Fc.

All the previous observations can be easily transposed in terms of hardness as a direct relationship exists with the ferrite ratio (Fig. 1c). As an example, Figures 2c,d highlight the specific finding that cutting a FP steel with a higher hardness can result in lower machining forces. The normal force Fn can be reduced by 46% by increasing the hardness from 140 to 260HB. To analyse this, one has to keep in mind that for a given rake angle, friction and work hardening are established as governing the machining forces via their influence on the chip thickness and shear angle [START_REF] Childs | Metal Machining: Theory and Application[END_REF]. Tribological experiments were thus performed to assess the friction contribution and extend the data already collected on some of the variants in a previous work [START_REF] Fabre | Optimization of surface roughness in broaching[END_REF].

As generally reported in metals [START_REF] Fabre | Optimization of surface roughness in broaching[END_REF][START_REF] Rech | Characterisation of friction and heat partition coefficients at the tool-work material interface in cutting[END_REF], the friction coefficient µapp decreased when the sliding velocity increased (Fig. 3). Moreover, µapp was seen to follow the same trends as machining forces when varying the ferrite ratio. From a quantitative point of view, at 2m/min, µapp increased by 20% when increasing the ferrite ratio from 5 to 70%. The results at 60m/min should be considered with caution as the pins exhibited a certain amount of wear which could explain the greater deviation noted in these tests. However, a reduction of 25% and 49% in terms of friction was identified at 2 and 60m/min respectively when the hardness was increased from 140 to 260HB. Decreasing F% enabled to limit the ploughing phenomenon but also to significantly reduce the adhesion tendency of the FP steels onto the friction pins.

The correlation between the trends identified in both the fundamental cutting and the tribological tests strongly suggests that when broaching a material with larger ferrite content, machining forces increase mainly due to a larger adhesion component, thus a tribological effect. Indeed, several studies showed that flow stress and then the machining forces increase when reducing the ferrite content [19][20]. This is contrary to what was observed in the present work tending to show that the friction contribution is predominant compared to the flow stress one. This is supported by the fact that the normal force Fn, component prone to be more sensitive to friction, was affected to a larger extent, a phenomenon also observed by [START_REF] Akasawa | Effect of microstructure and hardness on the machinability of medium-carbon chrome-molybdenum steel[END_REF].

Influence of a lubricant and TiN coating

To better highlight the influence of tribology, the contact conditions at the tool-material interface were modified by applying a lubricant and a TiN coating to both the tool and the friction pin. Ten variants from the 15 were selected and were cut at vc = 2m/min and h = 40µm, cutting conditions, which exhibited the largest variations when changing the FP microstructure. The following results are presented only in terms of normal force Fn, as this was shown to be the most sensitive to friction. Compared to the uncoated dry conditions, applying straight oil reduced the normal force by 35% at low ferrite ratio and up to around 60% for F% > 50% (Fig. 4a). This appears to confirm that tribological aspects are the main contributing factor to the cutting behaviour, as the oil is able to limit the higher adhesion tendency of the softer materials, thus leading to a stronger reduction of the machining forces.

During these tests, chips were also collected, embedded and polished to assess their average thickness t. The chip thickness ratio λh was then calculated as the ratio of t to h. Under dry conditions, λh was seen to drastically increase by a factor between 2 and 2.5 when increasing F% (Fig. 4b) or decreasing the hardness (Fig. 4e). Thicker chips are known to occur primarily when the friction along the tool-chip contact length is greater [START_REF] Childs | Metal Machining: Theory and Application[END_REF]. This trend is consistent with the increase generally observed in the normal force (Fig. 4a).

Similarly, λh was lowered by a factor close to 3 when using straight oil with steels with a large F%, attesting to its ability to lubricate the tool-material interface (Figs. 4b,e). The fact that the lubricant was straight oil and not a water-based emulsion excluded a potential thermal effect due to a beneficial cooling of the process. Nevertheless, although the lubricant significantly limited the effect of the FP microstructural parameters, it should be noted that the same trends as in dry broaching were found, but with a much lower amplitude.

Applying a TiN coating under lubricated conditions resulted in a further 35% reduction of Fn on average, and maintained the same trends as the uncoated conditions across the whole range of FP microstructures. However, Figures 4b,e show that no further modifications of λh were induced by this combination.

Figures 4c,f confirm a significant impact of straight oil on the tribological tests with an average reduction of µapp of 85% compared to dry conditions. Combining lubrication and the TiN coating did not improve the friction behaviour with µapp remaining at around 0.13 in average. This important reduction is in agreement with the decrease generally observed in the machining outputs and confirms the assumptions regarding the main contribution of friction in broaching.

Conclusions

This paper has presented a systematic study combining fundamental cutting tests and tribological experiments to identify the key parameters controlling the broaching performance of FP steels. It showed that the ferrite ratio F% had an important impact on the machining forces and chip formation with the main trends reported in Table 3. A strong correlation between the above and tribological testing was identified, and highly similar trends were observed regarding the apparent friction coefficient (Table 3), i.e. increasing F% led to increasing machining forces and apparent friction coefficient. This shows that broaching is a friction-driven process, and that tribology plays a major role in machining performance. Moreover, the relationship between FP microstructural parameters and the friction coefficient was identified, which is of definite interest to the community focusing on tribological aspects. Finally, a secondary outcome of this study suggests that tribological tests performed under relevant conditions could be used as a simplified method to assess the machining performance of FP steels in broaching.

Figure 1 .

 1 Figure 1. a) Examples of microstructures obtained when increasing the carbon content/ferrite ratio, b) heat treatment cycles applied to generate the various FP microstructures, c) correlation between ferrite ratio and macroscopic hardness and d,e) examples of corresponding micrographs when applying the heat treatment cycles to C45.

Figure 2 .

 2 Figure 2. Influence of ferrite ratio and macroscopic hardness on the cutting force Fc (a,c) and normal force Fn (b,d).

Figure 3 .

 3 Figure 3. Influence of ferrite ratio on the apparent friction coefficient when using uncoated friction pins under dry conditions.

Figure 4 .

 4 Figure 4. Influence of ferrite ratio and hardness on the normal force Fn (a,d), chip thickness ratio (b,e) and apparent friction coefficient (c,f) under dry and lubricated conditions.

Table 3

 3 Summary of the main influence of the investigated factors on the broaching and friction outputs.

	Type	Factor	Fc	Fn	λh	µapp
	FP	↑ F% (5-70%)	↑	↑	↑	↑
	parameters	↑ HB (140-260HB)	↓	↓	↓	↓
	Cutting	↑ Vc/ Vsl (2-60m•min -1 )	↓	↓	↓	↓
	conditions	↑ h (10-40 µm)	↑	↑	↑
	Contact conditions	Lub/TiN+Lub	↓	↓	↓	↓

Acknowledgements

This work has been carried out with a financial grant from the Research Fund for Coal and Steel (RFCS) of the European Community under the IMMAC project (grant agreement num. RFSR-CT-2014-00020).

The authors would also like to acknowledge the support of EKIN S. Coop. for providing the tools for the broaching tests as well as SIDENOR Basauri and ASCOMETAL-Creas for developing the specific heat treatments and providing the adequate steel variants.


Gulsev Ozen

Yasmine Amgoud

Heba Abdelazeem

Salma Mani

Chabha Benyahia

Amel Bouhadoun

Alexy Tran-Dinh

Yves Castier

Alice Guyard

Dan Longrois

Adam M Silverstein

Xavier Norel
email: xavier.norel@inserm.fr

Downregulation of PGI2 pathway in Pulmonary Hypertension Group-III patients

Keywords: Human pulmonary hypertension, pulmonary artery, PASMC, distal lung tissue, PGI2, prostacyclin synthase

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

Introduction

Pulmonary hypertension (PH) is characterized by a progressive increase in mean pulmonary artery pressure (mPAP>20 mmHg) and vascular resistance due to sustained pulmonary artery vasoconstriction [START_REF] Cogolludo | Mechanisms controlling vascular tone in pulmonary arterial hypertension: implications for vasodilator therapy[END_REF][START_REF] Simonneau | Haemodynamic definitions and updated clinical classification of pulmonary hypertension[END_REF]. The clinical classification of PH is intended to categorize multiple clinical conditions into five groups. Depending on this classification, PH Group-III is defined as PH due to lung diseases and/or hypoxia. Common lung diseases associated with PH are chronic obstructive pulmonary disease (COPD), interstitial lung disease and combined pulmonary fibrosis and emphysema [START_REF] Galie | ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT)[END_REF].

Most of the studies examining the pathogenesis of PH are performed in PH Group-I (idiopathic/heritable), which is more commonly referred to as Pulmonary Arterial Hypertension (PAH). In these patients, few studies have shown that the increased production of vasoconstrictor/proliferative mediators [such as endothelin-1 (ET-1), thromboxane (TxA2)] and reduced production of vasodilator/anti-proliferative mediators [such as prostacyclin (PGI2), nitric oxide (NO)] lead to pulmonary endothelial dysfunction and the development of PH [START_REF] Giaid | Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension[END_REF][START_REF] Adatia | Thromboxane A2 and prostacyclin biosynthesis in children and adolescents with pulmonary vascular disease[END_REF][START_REF] Christman | An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension[END_REF][START_REF] Yoshibayashi | Plasma endothelin concentrations in patients with pulmonary hypertension associated with congenital heart defects. Evidence for increased production of endothelin in pulmonary circulation[END_REF].

Among these mediators, more attention is focused on PGI2 because of the widespread use of it and its stable mimetics for treating PH. PGI2, a lipid mediator derived from arachidonic acid, is a powerful vasodilator and a potent inhibitor of platelet adhesion/aggregation and smooth muscle cell proliferation and growth. Cyclooxygenase (COX) catalyzes the conversion of arachidonic acid to prostaglandin H2 (PGH2), which is then converted to PGI2 by PGI2 synthase (PGIS), an enzyme constitutively expressed, among others, in pulmonary vascular endothelial cells [START_REF] Mahajan | Altered prostanoid metabolism contributes to impaired angiogenesis in persistent pulmonary hypertension in a fetal lamb model[END_REF][START_REF] Tuder | Prostacyclin synthase expression is decreased in lungs from patients with severe pulmonary hypertension[END_REF], smooth muscle cells [START_REF] Itoh | Effects of IL-1beta, TNF-alpha, and macrophage migration inhibitory factor on prostacyclin synthesis in rat pulmonary artery smooth muscle cells[END_REF], and bronchial epithelial cells [START_REF] Van Ly | Characterising the mechanism of airway smooth muscle beta2 adrenoceptor desensitization by rhinovirus infected bronchial epithelial cells[END_REF]. Once PGI2 is released from cells, it binds to the prostanoid IP receptor, a Gs-coupled receptor, and elevates cAMP levels [START_REF] Kawka | Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium[END_REF][START_REF] Parker | Effects of chronic estrogen-receptor blockade on ovine perinatal pulmonary circulation[END_REF][START_REF] Feletou | Endothelium-mediated control of vascular tone: COX-1 and COX-2 products[END_REF][START_REF] Clapp | The mechanistic basis of prostacyclin and its stable analogues in pulmonary arterial hypertension: Role of membrane versus nuclear receptors[END_REF]. These mechanisms are responsible for vasodilation in human lung [START_REF] Norel | Prostacyclin release and receptor activation: differential control of human pulmonary venous and arterial tone[END_REF].

Epoprostenol (PGI2) was the first drug approved by the FDA in 1984 for the treatment of PH [START_REF] Levarge | Prostanoid therapies in the management of pulmonary arterial hypertension[END_REF], and then other PGI2 analogues with better stability profiles, such as iloprost and treprostinil, were developed. PGI2 therapy improves the functional capacity and decreases the mortality rate of PH Group-I patients [START_REF] Lau | Epidemiology and treatment of pulmonary arterial hypertension[END_REF][START_REF] Simonneau | Treprostinil Study, Continuous subcutaneous infusion of treprostinil, a prostacyclin analogue, in patients with pulmonary arterial hypertension: a double-blind, randomized, placebo-controlled trial[END_REF]. Until now, these treatments have not been recommended for PH Group-III patients by European Guidelines due to the lack of randomized controlled trials [START_REF] Olschewski | Pulmonary hypertension due to lung diseases: Updated recommendations from the Cologne Consensus Conference 2018[END_REF].

The importance of PGIS in the development of PH has been demonstrated in several studies conducted in PH animal models and PH patients (mostly Group-I). However, there are differing results regarding PGIS expression and PGI2 production between PH and non-PH preparations [START_REF] Mahajan | Altered prostanoid metabolism contributes to impaired angiogenesis in persistent pulmonary hypertension in a fetal lamb model[END_REF][START_REF] Tuder | Prostacyclin synthase expression is decreased in lungs from patients with severe pulmonary hypertension[END_REF][START_REF] Badesch | Decreased arterial wall prostaglandin production in neonatal calves with severe chronic pulmonary hypertension[END_REF][START_REF] Blumberg | Increased pulmonary prostacyclin synthesis in rats with chronic hypoxic pulmonary hypertension[END_REF][START_REF] Fike | 2nd, eNOS and prostanoid enzymes in lungs of newborn piglets with chronic aortopulmonary shunts[END_REF][START_REF] Fike | Arachidonic acid metabolites and an early stage of pulmonary hypertension in chronically hypoxic newborn pigs[END_REF][START_REF] Jiang | Paracrine effects of bone marrowderived endothelial progenitor cells: cyclooxygenase-2/prostacyclin pathway in pulmonary arterial hypertension[END_REF][START_REF] Schroll | Effects of selective and unselective endothelin-receptor antagonists on prostacyclin synthase gene expression in experimental pulmonary hypertension[END_REF][START_REF] Stearman | Functional prostacyclin synthase promoter polymorphisms. Impact in pulmonary arterial hypertension[END_REF]. Many of the PH animal models have limitations, and some do not have complex arterial occlusive remodeling which is the characteristic of human PH diseases 4 [START_REF] Ryan | Rodent models of pulmonary hypertension: harmonisation with the world health organisation's categorisation of human PH[END_REF]. Therefore, studies conducted using PH patients are of great importance. To our knowledge, there are no studies evaluating the PGIS and PGI2 levels in PH Group-III patients, which is one of the most common and lethal form of PH [START_REF] Wijeratne | Increasing Incidence and Prevalence of World Health Organization Groups 1 to 4 Pulmonary Hypertension: A Population-Based Cohort Study in Ontario, Canada[END_REF].

The aim of this study is to determine whether PH Group-III is associated with alterations in the expression of PGIS and the formation of PGI2 in humans. To explore this hypothesis, we measured PGIS expression and PGI2 production in large variety of preparations from various locations in the pulmonary circulation, including human pulmonary artery (HPA), pulmonary vein (HPV), distal lung tissue, pulmonary artery smooth muscle cells (hPASMC), and bronchi in PH Group-III and control patients. Our results studying physiological alterations in PH Group-III patients provide insight into disease pathogenesis and its treatments.

Materials and Methods

Human pulmonary and bronchi preparations

Human pulmonary and bronchial preparations were collected in Bichat Hospital (Paris) after obtaining patients' informed consent with Ethics Committee approval from INSERM and AP-HP (CEERB du GHU Nord) Institutional Review Board (n° IRB00006477). These investigations conform to the principles outlined in the Declaration of Helsinki. Control preparations were obtained from patients (20 males, 4 females and 8 anonymous donors) who underwent surgery, mostly for lung carcinoma, while PH preparations were obtained from patients (26 males, 9 females) who had undergone surgery for lung transplantation. Patient categories are PH due to lung diseases and/or hypoxia [Group-III of PH classification [START_REF] Galie | ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT)[END_REF]], with detailed characteristics presented in supplemental data (Table S1), and control patient characteristics were presented in Table S2. PH lungs used in our study were from patients having catheter-measured mPAP ≥ 20 mmHg. All preparations were dissected in the anatomical pathology laboratory and used within 1-12 h post-surgery. HPA and HPV preparations (6-2 mm internal diameter) were dissected from pulmonary lobes. The "distal lung tissue" was cut at the periphery of the pulmonary lobes and contained vessels or bronchioles (<1 mm internal diameter) and parenchyma.

Culture of human pulmonary artery smooth muscle cells

The culture of hPASMC was carried out in a biosafety level 2 laboratory in a vertical laminar flow hood. The hPASMC were obtained from pulmonary arteries derived from control and PH Group-III patients. These arteries were removed after dissection of lung lobes or whole lungs. First, proximal arteries were opened, cleaned of any connective tissue (parenchyma) and then rinsed with phosphate-buffered saline (PBS) containing 1/20 penicillin, streptomycin, amphotericin B (PSA). After rinsing, the artery media was isolated and cut with a scalpel into small 1-2 mm pieces. This preparation was placed in a medium containing collagenase (Type 1) and elastase and then incubated for 30 to 40 minutes at 37°C. After incubation and enzymatic digestion of the extracellular matrix, the preparation was filtered (40 microns filter) and centrifuged at 1000 rpm for 10 minutes at 20°C. After centrifugation, the supernatant was aspirated, and the pellet was resuspended in a T25 flask containing Smooth Muscle Cell Growth Medium 2 supplemented with 20% fetal calf serum (FCS), PSA and growth factors [human EGF (epidermal growth factor), human bFGF (fibroblast growth factor), IGF (insulin-like growth factor)] to allow the proliferation of smooth muscle cells (SMC). The cells were cultured in an incubator at 37°C in humid atmosphere containing 5% CO2. When confluence was 6 reached, the hPASMC were detached from the T25 flask using 2 ml trypsin. In this first passage, the cells were resuspended in 12 ml of culture medium (10% FCS) and transferred to a T75 flask. Depending on the extent of cell confluence, passages were performed approximately every 2 weeks. The SMC were confirmed morphologically; we obtained spindle shaped cells forming the "hill and valley" configuration which is typical of SMC (see Figure 3). In passage 3-4, the hPASMC were washed twice with PBS. Subsequently, hPASMC were detached as described earlier and diluted in culture medium with 10% FCS in order to have a concentration of 3x10 6 cells per 100 ml. For one cell culture derived from one individual, the cells in homogeneous suspension were then seeded (2 mL /well) in 6 well plates. After proliferation of the cells in the 6-well plates (90% confluence), the culture medium (10% FCS) was aspirated and replaced with a 0 % FCS culture medium. Cells were then kept in an incubator at 37°C in a humid atmosphere containing 5% CO2 for 48 h.

Western blot analysis

Human pulmonary arteries, veins, bronchi, and distal lung tissue samples were homogenized in liquid nitrogen, using a porcelain mortar. Homogenates were diluted (100 mg/ml) in lysis buffer (RIPA buffer supplemented with protease and phosphatase inhibitors). hPASMC in 6 well plates were washed twice with PBS and lysed with lysis buffer. Proteins were quantified by BCA protein assay kit, and a 12.5-50 μg of protein sample were loaded on a 12% polyacrylamide gel. Proteins were electroblotted onto nitrocellulose membranes. Membranes were blocked (TBS, 0.05% Tween 20, 5% non-fat dry milk) and incubated overnight at 4°C with a primary antibody specifically targeting PGIS in TBS (0.05%) Tween-20 (1%) non-fat dry milk. Subsequently, the membranes were incubated with an appropriate peroxidaseconjugated secondary antibody. Bands were visualized using the ECL prime luminescence system. For quantification, the film was scanned, and the integrated optical density of the bands was estimated with Scion Image (Scion Corporation, NIH, Frederick, MD, USA) and normalized to β-actin. For densitometrical analysis, no brightness/contrast adjustments have been performed, except in Figure 3, where automatic contrast adjustments were performed on the area (about 2cm x 2cm) around the band of interest, prior to blank and band density calculations. For PGIS western blot bands, the inferior band corresponding to 56 kDa was quantified.

ELISA measurements

Samples of pulmonary arteries, pulmonary veins and distal lung tissue were dissected and cut into small pieces and placed into 12-well plates (100-200 mg tissue/well) containing RPMI (pH 7.4) supplemented with antibiotics (penicillin, 1000 IU/mL; streptomycin, 100 μg/mL) and an antimycotic agent (amphotericin, 0.25 μg/mL) for 12 hours. The volume of the RPMI was adjusted to 1 mL for 70 mg of tissue. In addition, hPASMC in 6 well plates were incubated with culture medium for 48 hours. All cell/tissue incubations were conducted at 37°C in a humidified atmosphere of 5% CO2 in air using a culture incubator. 6-keto-PGF1α concentrations were measured in supernatants/culture medium from incubation of pulmonary arteries, pulmonary veins, distal lung tissue and hPASMC. For bronchi, 6-keto-PGF1α levels were measured in tissue homogenates. All measurements were conducted using an enzyme immunoassay kit.

Statistical analysis

All results obtained from different patients (n) were expressed as mean ± standard error of the mean (SEM). After testing normality and equal variances of the data, we performed Student or Mann Whitney-Rank Sum tests and calculated Pearson or Spearman correlation coefficients (r). For all statistical tests, P-values less than 0.05 were considered statistically significant. Statistical analyses were performed using SigmaStat version 3.5 (Systat Software, Point Richmond, CA, USA). ELISA measurements were performed in duplicate and an average taken in each sample to calculate the final mean data.

Compounds and materials

Protease inhibitor cocktail, antibiotics, antimycotic, and β-actin antibody were from Sigma-Aldrich (St. Louis, MO, USA). 6-keto PGF1α ELISA kit and antibody against PGIS were from Cayman Chemical (Ann Arbor, MI, USA; Reference number: 515211, 160640 respectively) [START_REF] Ozen | Inhibition of microsomal PGE synthase-1 reduces human vascular tone by increasing PGI2 : a safer alternative to COX-2 inhibition[END_REF][START_REF] Ozen | Bronchodilation induced by PGE2 is impaired in Group III pulmonary hypertension[END_REF]. Polyclonal rabbit antibody used as secondary antibody was from Jackson ImmunoResearch 73120, (Pensylvannia, USA). RPMI, trypsin and collagenase were from Gibco Invitrogen (Paisley, UK). Elastase was from Worthington (Lakewood, NJ, USA). BCA protein assay kit was from Thermo (Rockford, USA). Nitrocellulose membranes and ECL Plus® system were from Amersham Biosciences (Buckinghamshire, UK). Smooth Muscle Cell Growth Medium 2 was from PromoCell (Heidelberg, Germany). Phosphatase inhibitor (phosSTOP) was from Roche (Basel, Switzerland)).

Results

PGIS expression and 6-keto-PGF1α levels in human pulmonary arteries derived from control and PH Group-III patients

The expression of PGIS enzyme was significantly decreased in HPA preparations derived from PH patients versus control patients (Figures 1A, 1C, P=0.02). Consistent with this finding, 6-keto-PGF1α levels were significantly lower in HPA preparations derived from PH patients as compared to control patients (Figure 1B, P<0.001).

PGIS expression and 6-keto-PGF1α levels in human pulmonary veins derived from control and PH Group-III patients

The expression of PGIS enzyme was not significantly different between HPV preparations derived from PH patients and control patients (Figures 2A,2C, P=0.89).

Consistent with this finding, 6-keto-PGF1α levels, while decreased, were not statistically significantly different between HPV preparations derived from PH patients and control patients (Figure 2B, P=0.18).

PGIS expression and 6-keto-PGF1α levels in human pulmonary artery smooth muscle cells derived from control and PH Group-III patients

The expression of PGIS enzyme was significantly decreased in hPASMC derived from PH patients versus control patients (Figures 3A, 3C, P=0.004). Consistent with this finding, 6keto-PGF1α levels were significantly lower in hPASMC derived from PH patients as compared to control patients (Figure 3B, P=0.008). Furthermore, the production of 6-keto-PGF1α from these cells positively correlated with PGIS expression (Figure 3D) and was inversely correlated with mPAP (Figure 3E).

PGIS expression and 6-keto-PGF1α levels in distal lung tissue derived from control and PH Group-III patients

The expression of PGIS enzyme was not significantly different in distal lung tissue derived from PH patients versus control patients (Figures 4A,4C; P=0.27); whereas, the 6-keto-PGF1α levels were significantly lower in distal lung tissue derived from PH patients as compared to control patients (Figure 4B, P=0.03).

PGIS expression and 6-keto-PGF1α levels in human bronchi derived from control and PH Group-III patients

The levels of PGIS enzyme expression and 6-keto-PGF1α were similar in human bronchi derived from PH patients and control patients (Figures 5A, 5B, 5C; P=0.91 P=0.37 respectively).

Discussion and Conclusions

The current study demonstrates for the first time that the expression of PGIS and/or 6keto-PGF1α levels are significantly decreased in human pulmonary arteries, hPASMC, and distal lung tissue derived from PH Group-III patients versus control patients. On the other hand, human pulmonary veins and bronchi preparations showed no differences. These results suggest that the deficiency of the PGIS pathway is restricted to pulmonary arteries.

The therapeutic importance of PGI2 therapy in PH may be related to the deficiency of PGIS in pulmonary tissue, however this has not been investigated in PH Group-III patients. In the present study, the significant decrease of PGIS expressions in arterial preparations derived from PH Group-III patients (Figures 1A and3A) is consistent with previous immunohistochemical results from Tuder et al. These authors showed a lower PGIS expression in small/medium-size, but not in large, pulmonary arteries derived from PH Group-I patients [START_REF] Tuder | Prostacyclin synthase expression is decreased in lungs from patients with severe pulmonary hypertension[END_REF]. Similarly, studies performed on PH rats, lambs or piglets demonstrated that PGIS expression was significantly decreased in the pulmonary arteries [START_REF] Mahajan | Altered prostanoid metabolism contributes to impaired angiogenesis in persistent pulmonary hypertension in a fetal lamb model[END_REF][START_REF] Fike | Arachidonic acid metabolites and an early stage of pulmonary hypertension in chronically hypoxic newborn pigs[END_REF][START_REF] Jiang | Paracrine effects of bone marrowderived endothelial progenitor cells: cyclooxygenase-2/prostacyclin pathway in pulmonary arterial hypertension[END_REF]. In accordance, PGIS overexpression/gene transfer in animal models protected against PH [START_REF] Geraci | Pulmonary prostacyclin synthase overexpression in transgenic mice protects against development of hypoxic pulmonary hypertension[END_REF][START_REF] Nagaya | Gene transfer of human prostacyclin synthase ameliorates monocrotaline-induced pulmonary hypertension in rats[END_REF]. It is worth mentioning that decreased PGIS expression not only contributes to the pathophysiology of PH by reducing PGI2 production but likely also by shifting the PGH2 metabolism towards TxA2.

The latter effect was demonstrated by increased TxA2 levels in urine or plasma derived from PH patients [START_REF] Adatia | Thromboxane A2 and prostacyclin biosynthesis in children and adolescents with pulmonary vascular disease[END_REF][START_REF] Christman | An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension[END_REF][START_REF] Zheng | Changes of thromboxane A2 (TXA2) and prostacyclin (PGI2) in COPD patients with pulmonary hypertension[END_REF][START_REF] Fuse | Plasma thromboxane B2 concentration in pulmonary hypertension associated with congenital heart disease[END_REF]. This shift towards TxA2 may lead to vasoconstriction, an increase of SMC proliferation and matrix protein synthesis, an exaggerated response to growth factors, and endothelial dysfunction. Taken altogether, the level of PGIS expression is a key factor in PH development. [START_REF] Geraci | Pulmonary prostacyclin synthase overexpression in transgenic mice protects against development of hypoxic pulmonary hypertension[END_REF][START_REF] Nagaya | Gene transfer of human prostacyclin synthase ameliorates monocrotaline-induced pulmonary hypertension in rats[END_REF].

Our results showed no change in PGIS expression in distal lung tissue derived from PH Group-III patients (Figure 4A), an observation that is consistent with previous findings in whole lung homogenates derived from pigs with elevated pulmonary blood flow and PH [START_REF] Fike | 2nd, eNOS and prostanoid enzymes in lungs of newborn piglets with chronic aortopulmonary shunts[END_REF]. In contrast, studies using whole lung homogenates from chronic hypoxia or monocrotaline (MCT)-induced PH rats showed an increased PGIS expression [START_REF] Blumberg | Increased pulmonary prostacyclin synthesis in rats with chronic hypoxic pulmonary hypertension[END_REF][START_REF] Schroll | Effects of selective and unselective endothelin-receptor antagonists on prostacyclin synthase gene expression in experimental pulmonary hypertension[END_REF]. Consistent with this finding, PGIS mRNA expression in human whole lung tissue derived from PH Group-I patients showed a trend toward a positive correlation with mPAP [START_REF] Stearman | Functional prostacyclin synthase promoter polymorphisms. Impact in pulmonary arterial hypertension[END_REF]. When considering these sometimes consistent and sometimes contradictory observations in lung tissue, it appears that the changes in endogenous PGIS expression and activity may be dependent on the samples analyzed (pulmonary artery versus whole lung) or the model/ PH group used. 10 Previous studies using PH animal models demonstrated that decreased PGIS expression was associated with lower PGI2 production in pulmonary arterial preparations [START_REF] Mahajan | Altered prostanoid metabolism contributes to impaired angiogenesis in persistent pulmonary hypertension in a fetal lamb model[END_REF][START_REF] Badesch | Decreased arterial wall prostaglandin production in neonatal calves with severe chronic pulmonary hypertension[END_REF][START_REF] Fike | Arachidonic acid metabolites and an early stage of pulmonary hypertension in chronically hypoxic newborn pigs[END_REF][START_REF] Jiang | Paracrine effects of bone marrowderived endothelial progenitor cells: cyclooxygenase-2/prostacyclin pathway in pulmonary arterial hypertension[END_REF]. This is consistent with our current study in human pulmonary artery (Figure 1A andB) and even more so in hPASMC where we show a significant positive correlation between PGIS expression and 6-keto-PGF1α levels (Figure 3D). In addition, our results demonstrated a significant inverse correlation between mPAP and the PGI2 metabolite released by hPASMC of the same patients (Figure 3E), a result suggesting that PGI2 levels are an important determinant for the severity of disease. Consistent with our results, 24h urinary excretion of a stable metabolite of PGI2, 2,3-dinor-6-keto-PGF1α, was reduced in patients with primary and secondary PH [START_REF] Christman | An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension[END_REF]. Our results from HPA could not be explained solely by the endothelial dysfunction observed in PH. In our previous work using the same fresh "control" pulmonary arteries, the production of PGI2 induced by arachidonic acid was significantly reduced by 68% and 56% in endothelium-stripped HPA and HPV, respectively [START_REF] Norel | Prostacyclin release and receptor activation: differential control of human pulmonary venous and arterial tone[END_REF]. This previous work and current data demonstrating attenuated PGI2 synthesis in PH hPASMC (Figure 3B) suggest that both endothelial and smooth muscle cells dysfunction are responsible for the decreased PGI2 levels measured in HPA in PH patients (Figure 1B). Furthermore, published work performed in PH Group-I patients [START_REF] Lai | Role of the prostanoid EP4 receptor in iloprostmediated vasodilatation in pulmonary hypertension[END_REF][START_REF] Fan | Mechanism of Beraprost Effects on Pulmonary Hypertension: Contribution of Cross-Binding to PGE2 Receptor 4 and Modulation of O2 Sensitive Voltage-Gated K(+) Channels[END_REF][START_REF] Falcetti | Smooth muscle proliferation and role of the prostacyclin (IP) receptor in idiopathic pulmonary arterial hypertension[END_REF] and our recent studies in PH Group-III patients [START_REF] Ozen | Bronchodilation induced by PGE2 is impaired in Group III pulmonary hypertension[END_REF] demonstrated that IP receptor expression was also decreased in pulmonary preparations. These data support the whole PGI2 pathway plays a role in the development of PH. Importantly, our data contribute to understanding the beneficial role of PGI2-mimetic treatments in PH Group-III patients (in particular with severe PH) that were previously suggested [START_REF] Dernaika | Iloprost improves gas exchange and exercise tolerance in patients with pulmonary hypertension and chronic obstructive pulmonary disease[END_REF][START_REF] Faria-Urbina | Inhaled Treprostinil in Pulmonary Hypertension Associated with Lung Disease[END_REF][START_REF] Hegewald | Sustained improvement with iloprost in a COPD patient with severe pulmonary hypertension[END_REF][START_REF] Reichenberger | Effects of nebulised iloprost on pulmonary function and gas exchange in severe pulmonary hypertension[END_REF][START_REF] Olschewski | Inhaled prostacyclin and iloprost in severe pulmonary hypertension secondary to lung fibrosis[END_REF][START_REF] Wang | Hemodynamic and gas exchange effects of inhaled iloprost in patients with COPD and pulmonary hypertension[END_REF][START_REF] Harari | Pulmonary Hypertension in Parenchymal Lung Diseases: Any Future for New Therapies?[END_REF].

Interestingly, in PH distal lung tissue preparations, although there was no significant change in PGIS expression, 6-keto-PGF1α production was significantly attenuated (Figure 4B).

One mechanism that could explain this discrepancy is the tyrosine nitration of PGIS by oxidative stress and/or inflammation, which can be observed in PH. Nitration of PGIS could result in its inactivation, an effect that was described in pulmonary artery endothelial cells derived from a model of persistent PH in new-born lambs [START_REF] Mahajan | Altered prostanoid metabolism contributes to impaired angiogenesis in persistent pulmonary hypertension in a fetal lamb model[END_REF]. This discrepancy between PGIS expression and PGI2 levels in distal lung tissue from PH Group-III patients should be further investigated. In addition, the level of 6-keto-PGF1α released in supernatants from distal lung tissue was approximately 10-fold less when compared with supernatants from artery or vein preparations, an observation that may be explained by the complex structure of distal lung tissue which could trap the PGI2 metabolite.

Although it has been suggested that pulmonary veins and bronchi [START_REF] Ozen | Bronchodilation induced by PGE2 is impaired in Group III pulmonary hypertension[END_REF] play an important role in normal lung physiology and the pathophysiology of PH, there is a lack of studies exploring the prevalence of venous/bronchial dysfunction in the various forms of PH. In our study, dysregulation of the PGIS expression and activity was not observed in either pulmonary vein (Figure 2) or bronchi (Figure 5) derived from PH Group-III patients. Consistent with our results, Badesh et al. demonstrated that PGI2 release was not changed in pulmonary veins derived from calves with hypoxia-induced severe PH. The authors suggested that pulmonary veins are not exposed to increased wall tension or shear stress and are protected from the hemodynamic stress of PH [START_REF] Badesch | Decreased arterial wall prostaglandin production in neonatal calves with severe chronic pulmonary hypertension[END_REF].

Most of our knowledge about PH and the development of pharmacological/therapeutic strategies come from studies using PH animal models. Unfortunately, most of these models do not fully mirror the pathophysiological features of this disease. For instance, chronic hypoxia is defined as a model of PH Group-III; however, it is species-dependent and is not reflective of the challenges that exist for improving the vascular bed, haemodynamic status or mortality rate in humans [START_REF] Ryan | Rodent models of pulmonary hypertension: harmonisation with the world health organisation's categorisation of human PH[END_REF]. For all of these reasons, in vitro studies using samples isolated from PH patients, especially Group-III which is one of the most common and lethal form of PH [START_REF] Wijeratne | Increasing Incidence and Prevalence of World Health Organization Groups 1 to 4 Pulmonary Hypertension: A Population-Based Cohort Study in Ontario, Canada[END_REF], are indispensable.

While these data are compelling, there are some limitations for these types of assessments. Obtaining fresh lung samples after lung transplantation of PH Group-III is quite difficult, and while the n value of experiments is acceptable for statistical and scientific interpretation, it is not robust. This low availability of tissue also hindered assessing the cellular localization of PGIS by immunohistochemistry in these studies. In the literature, two studies have described PGIS localization in human pulmonary endothelial [START_REF] Tuder | Prostacyclin synthase expression is decreased in lungs from patients with severe pulmonary hypertension[END_REF] and epithelial cells [START_REF] Van Ly | Characterising the mechanism of airway smooth muscle beta2 adrenoceptor desensitization by rhinovirus infected bronchial epithelial cells[END_REF].

Our results demonstrate that PGIS is expressed in hPASMC, an observation only previously described in rat PASMC [START_REF] Itoh | Effects of IL-1beta, TNF-alpha, and macrophage migration inhibitory factor on prostacyclin synthesis in rat pulmonary artery smooth muscle cells[END_REF]. Further experiments are needed to evaluate PGIS expression in fibroblasts or alveolar type I/II cells in control and PH patients.

Conclusion

Our results demonstrate significant decreases of PGIS expression and/or 6-keto-PGF1α levels in human pulmonary artery, smooth muscle cells, and distal lung tissue derived from PH Group-III patients. More importantly, our study suggests for the first time that PH Group-I and Group-III share a common pathophysiological decrease of PGIS pathway expression and function. Despite the fact that inhaled PGI2 therapy in PH Group-III patients seems to be a promising treatment [START_REF] Dernaika | Iloprost improves gas exchange and exercise tolerance in patients with pulmonary hypertension and chronic obstructive pulmonary disease[END_REF][START_REF] Faria-Urbina | Inhaled Treprostinil in Pulmonary Hypertension Associated with Lung Disease[END_REF][START_REF] Hegewald | Sustained improvement with iloprost in a COPD patient with severe pulmonary hypertension[END_REF][START_REF] Reichenberger | Effects of nebulised iloprost on pulmonary function and gas exchange in severe pulmonary hypertension[END_REF][START_REF] Olschewski | Inhaled prostacyclin and iloprost in severe pulmonary hypertension secondary to lung fibrosis[END_REF][START_REF] Wang | Hemodynamic and gas exchange effects of inhaled iloprost in patients with COPD and pulmonary hypertension[END_REF][START_REF] Harari | Pulmonary Hypertension in Parenchymal Lung Diseases: Any Future for New Therapies?[END_REF], current guidelines for PH management indicated that published experience with PGI2 therapy is scarce in PH Group-III, and it is not currently recommended for these patients [START_REF] Galie | ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT)[END_REF]. Our findings provide additional support for considering the use of PGI2 mimetics to treat PH Group-III.

Figure 1 A B C

 1B Figure 1

Figure 2 A B C

 2B Figure 2 A B

  Figure 3 A B

  Figure 4 A B

Figure 5 A B C

 5B Figure 5 A B

12

Acknowledgement

We would like to thank the Anapathology laboratory and the secretary of the Department of Anaesthesia and Intensive Care, CHU X. Bichat for their help. We would like to thank United Therapeutics for an educational grant supporting Yasmine Amgoud thesis and the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No: 665850 supporting Heba Abdelazeem thesis.