PhD Cara Verdellen
email: c.verdellen@hsk.nl

Jolande Van De Griendt

Andreas Hartmann

Tara Murphy

Psychological Treatment Guidelines TS 1 European clinical guidelines for Tourette Syndrome and other tic disorders. Part III: Behavioural and psychosocial interventions

Keywords:

Objective: This clinical guideline provides recommendations for the behavioural and psychosocial interventions (BPI) of children and adolescents with tic disorders prepared by a working group of the European Society for the Study of Tourette Syndrome (ESSTS). Method: A systematic literature search was conducted to obtain an update on the efficacy of BPI for tics. Relevant studies were identified using computerized searches of the Medline and PsycINFO databases and the Cochrane Library for the years 1950-2010. The search identified no meta-analyses, yet twelve (systematic) reviews and eight randomized controlled trials provided evidence for the current review. Results: Most evidence was found for habit reversal training (HRT) and the available but smaller evidence also supports the efficacy of exposure with response prevention (ERP). Both interventions are considered first line behavioural treatments for tics for both children and adults and should be offered to a patient, taking into account his preference. Treatments that are considered second line or add-on behavioural treatments are contingency management, function based interventions and relaxation training. Neurofeedback is still experimental. Almost no research was identified that examined the efficacy of psychosocial interventions, e.g., psychoeducation and group work. Conclusion: Based on clinical practice, this guideline recommends behavioural treatment as first line offer to patients in most cases. It should be embedded within a psychoeducational and supportive context and can be combined with drug treatment.

Introduction

Tic disorders (e.g., chronic motor or vocal tic disorder and Tourette syndrome; TS) are a common developmental disorder among children and adolescents. About 10% of children have (motor and/or vocal) tics during childhood, which in many cases disappear. However, approximately 1% of children and adolescents develop TS [START_REF] Robertson | The prevalence and epidemiology of Gilles de la Tourette syndrome. Part 1: the epidemiological and prevalence studies[END_REF], a condition that is characterized by multiple motor and vocal tics. Often, symptoms of Attention Deficit Hyperactivity Disorder (ADHD), Obsessive Compulsive Disorder (OCD) and Pervasive Developmental Disorder (PDD) coexist. Tics may vary in their severity and in the inconvenience they cause to an individual and his environment. Although not all children and families seek help [START_REF] Schlander | Tic disorders: Administrative prevalence and co-occurrence with attention-deficit/hyperactivity disorder in a German community sample[END_REF], many require some form of treatment or support to manage their tics and impaired social, emotional and behavioural functioning. The European Society for the Study of Tourette Syndrome (ESSTS) met in November 2008 and in August 2009 to discuss the development of guidelines for the treatment of tic disorders. The present guideline describes recommendations for the behavioural and psychosocial interventions (BPI) of tics in children and adolescents. The behavioural treatment of comorbidity in tic disorders is beyond the scope of this guideline, but some information can be found in Döpfner and Rothenberger [START_REF] Döpfner | Behavior therapy in tic-disorders with co-existing ADHD[END_REF], Döpfner and Rothenberger [START_REF] Döpfner | Tic-und Zwangsstörungen[END_REF] and Rothenberger et al. [START_REF] Rothenberger | Habit formation in Tourette Syndrome with associated obsessive-compulsive behaviour: At the crossroads of neurobiological modelling[END_REF].

Method

To obtain an update on scientific research on the efficacy and effectiveness of BPI for tic disorders, a literature search was performed on behavioural and psychosocial interventions using computerized searches of the Medline (PubMed) and PsycINFO databases (1950( -May 2010)). This search was performed using relevant terms (e.g., tics, tic disorder, Tourette's syndrome, behavio(u)r therapy, habit reversal, exposure and response prevention, relaxation, psychoeducation, support group, coping strategies). 'Map term to subject heading', in which the best-matching subject headings associated with a term are included, was used. In addition, the reference lists of the (review) articles identified through Medline and PsychINFO were reviewed for additional studies. The search produced an initial list of 159 publications, which revealed no meta-analyses, twelve (systematic) reviews, eight randomized controlled trials (RCT's) and one non-randomized controlled trial. Furthermore, multiple case studies were found. No studies were found on tic disorders comparing behaviour therapy with pharmacotherapy. The Cochrane library was also consulted and contained no information on BPI for tic disorders. Although the publication list comprises many relevant articles related to the subject, it should be noted that it is not exhaustive.

Results

Behavioural interventions

The most rigorously researched of all psychological interventions are behavioural treatments.

Overall, eight different behavioural treatments for tics were identified in the literature: massed (negative) practice (MP), habit reversal (HR), self-monitoring (SM), contingency management (CM)/function based interventions (FBI), relaxation training (RT)/hypnosis, exposure and response prevention (ER), cognitive-behavioural treatment (CBT) and bio(neuro)feedback (NF). Most studies utilized single-case experimental designs, using a multiple baseline design, a waiting list design (WL) or an alternating treatments design. Nine studies employed group-based methodology, eight of these with random assignment to treatment conditions. RCT's differed from one another on several variables (e.g., tic disorder, HR ingredients, sample age, outcome measures, duration of follow-up). Four studies included both children and adults, but only two studies consisted of children (age 7-18) only. Table 1 provides an overview of the RCT's of tic disorders.

---------------Include Table 1 here ------------------

Massed (negative) practice (MP)

MP is the oldest behavioural treatment for tics reported in the literature. MP involves the repeated, rapid, voluntary and effortful performance of tics for a specified period of time (e.g., 30 minutes), interspersed with brief periods of rest. The majority of single case studies into MP failed to find a decrease in tic frequency. In the one RCT comparing MP to HR in both children and adults with TS, MP failed to produce reductions in tics comparable to those produced by the HR condition [START_REF] Azrin | Habit reversal vs negative practice treatment of nervous tics[END_REF] (see Table 1). Overall, it can be concluded that MP has limited therapeutic value for tic disorders.

Habit Reversal (HR)

HR is the most extensively researched behavioural treatment for tics. It offers a set of techniques that help the patient become aware of the occurrence of a tic, followed by a socalled competing response training to interrupt or inhibit the tic [START_REF] Azrin | Habit reversal: A method of eliminating nervous habits and tics[END_REF][START_REF] Azrin | Treatment of Tourette Syndrome by habit reversal: A waiting-list control group comparison[END_REF][START_REF] Woods | Habit reversal: a review of applications and variations[END_REF]. HR may also comprise relaxation training, contingency management, and generalization training. Awareness of the tics is enhanced through response description, response detection, an early warning procedure, and situation awareness training. With competing response training the patient learns to initiate a response for 1 to 3 minutes, or until the urge goes away, contingent upon the urge to perform a tic or after the actual occurrence of the tic. Several dozens of uncontrolled studies into HR report tic reductions of 30 to 100%. Most studies involved small patient samples with unspecified diagnostic criteria. However, eight RCT's (see Table 1) were performed into HR indicating its' efficacy. Four studies were performed on adults only (age 18-60) [START_REF] Wilhelm | Habit reversal versus supportive psychotherapy for Tourette's disorder: a randomized controlled trial[END_REF][START_REF] Deckersbach | Habit reversal versus supportive psychotherapy in Tourette's disorder: a randomized controlled trial and predictors of treatment response[END_REF][START_REF] O'connor | A comparison of a behavioural and a cognitivebehavioural approach to the management of chronic tic disorders[END_REF][START_REF] O'connor | Evaluation of a cognitive-behavioural program for the management of chronic tic and habit disorders[END_REF], 1 study (the largest study up to date, N=126) included children only (age 9-17) [START_REF] Piacentini | Behavior therapy for children with Tourette disorder: a randomized controlled trial[END_REF] and 3 studies included both children and adults (age 6-62) [START_REF] Azrin | Habit reversal vs negative practice treatment of nervous tics[END_REF][START_REF] Azrin | Treatment of Tourette Syndrome by habit reversal: A waiting-list control group comparison[END_REF][START_REF] Verdellen | Exposure with response prevention versus habit reversal in Tourettes's syndrome: a controlled study[END_REF]. Most studies consisted of 10 to 14 weekly sessions [START_REF] Wilhelm | Habit reversal versus supportive psychotherapy for Tourette's disorder: a randomized controlled trial[END_REF][START_REF] Deckersbach | Habit reversal versus supportive psychotherapy in Tourette's disorder: a randomized controlled trial and predictors of treatment response[END_REF][START_REF] O'connor | A comparison of a behavioural and a cognitivebehavioural approach to the management of chronic tic disorders[END_REF][START_REF] O'connor | Evaluation of a cognitive-behavioural program for the management of chronic tic and habit disorders[END_REF][START_REF] Verdellen | Exposure with response prevention versus habit reversal in Tourettes's syndrome: a controlled study[END_REF]. Some studies spreaded the sessions over time after starting with weekly sessions, e.g. biweekly or monthly sessions. Follow-up data of at least 10 months duration showed maintenance of results. More information can be found in table 1. In addition to the RCT's, several review articles support evidence for HR [START_REF] Carr | Habit reversal treatment of tic disorders: a methodological critique of the literature[END_REF][START_REF] Cook | Evidence-based psychosocial treatments for tic disorders[END_REF][START_REF] Himle | Brief review of habit reversal training for Tourette syndrome[END_REF][START_REF] Peterson | Psychosocial management of tics and intentional repetitive behaviors associated with Tourette syndrome[END_REF][START_REF] Piacentini | Behavioral treatments for Tourette syndrome and tic disorders: state of the art[END_REF][START_REF] Piacentini | Habit reversal training for tic disorders in children and adolescents[END_REF]. Awareness training and competing response training seem to be the active components of the multi-component HR package that are necessary and sufficient for tic reduction [START_REF] Miltenberger | Habit reversal with muscle tics: Replication and component analysis[END_REF]. In summary, studies indicate that HR is effective for both vocal and motor tics, for children as well as adults, for patients receiving TS medications as well as those not doing so, for tic severity as well as tic frequency, and with no evidence of symptom substitution [START_REF] Woods | Barriers to dissemination: Exploring the criticisms of behavior therapy for tics[END_REF].

Self-monitoring (SM)

SM consists of having the patient record tics during a specified period by using a counter or a notebook. The goal is to identify when and in which situation a tic occurs. SM is often applied in multi-component treatment (HR) packages. Only a few case studies have been conducted in which SM was used as the primary treatment procedure, indicating that SM generally results in temporary improvement [START_REF] Peterson | An evaluation of behavioral treatments for Tourette syndrome[END_REF]. Immediate, rather than delayed recording of tics appears to be essential for tic reduction [START_REF] Billings | Self-monitoring in the treatment of tics: A single-subject analysis[END_REF][START_REF] Turpin | Effects of massed practice and cue-controlled relaxation on tic frequency in Gilles de la Tourette's syndrome[END_REF]. In summary, there is some support for the usefulness of SM, although the generalizability and durability of treatment gains remain unclear.

Contingency management (CM)/Function based interventions (FBI)

CM consists of the manipulation of environmental contingencies, so that tic-free intervals are positively reinforced and tic behaviours are ignored. Using tokens to reinforce the absence of tics is an example of a CM-based intervention [27]. CM is mostly used in multi-component treatment packages, making it difficult to assess the unique value of this specific technique. A more specialized type of CM is known as 'function based interventions' (FBI). In FBI, factors specific to the individual's unique environment are indentified which increase or decrease tics. The factors are altered to provide tic reductions. The effect of this intervention is reported in single case studies [START_REF] Watson | Brief functional analysis and treatment of a vocal tic[END_REF].

Relaxation training (RT)

The use of RT in the treatment of tics is based on the observation that increases in stress and anxiety result in concomitant increases in tic performance. Hence, tic reduction in RT might work indirectly via stress reduction. Relaxation can help reduce muscular tension. RT includes deep breathing, progressive muscle training, and imagery, and is mostly applied as part of a multi-component treatment. Two studies examined RT as a mono-component treatment under controlled conditions. Peterson and Azrin [START_REF] Peterson | An evaluation of behavioral treatments for Tourette syndrome[END_REF] found that RT resulted in an average tic decreases of 32% (N=6), which was less than the effect of competing response training (55% decrease) or SM (45% decrease). Bergin et al. [START_REF] Bergin | Relaxation therapy in Tourette syndrome: a pilot study[END_REF] conducted a controlled, randomized trial in children (age 7-18) comparing RT with minimal control therapy. After six weekly sessions, they found no differences between groups in terms of tic symptom severity (see Table 1). Improvements in tic symptoms were only short-term. In summary, it can be concluded that RT is not an evidence based treatment for tics, although it may have some merit in the short term. Hypnosis and cue-controlled relaxation, more specific forms of relaxation, have been shown useful in some cases [START_REF] Culbertson | A four-step hypnotherapy model for Gilles de la Tourette's syndrome[END_REF][START_REF] Hoogduin | Exposure, responspreventie en zelfcontrole bij Gilles de la Tourette-patiënten [Exposure, response prevention and self-control in patients with Gilles de la Tourette syndrome[END_REF] and need further study.

Exposure and response prevention (ER)

The application of ER to reduce tics is based on the association of unpleasant premonitory sensations followed by a motor or vocal tic that relieves the sensation [START_REF] Leckman | Premonitory urges in Tourette's syndrome[END_REF]. In learning theory terms, tics can be viewed as conditioned responses to premonitory, interoceptive stimuli. When such stimuli reoccur over time, the power of the associative interaction between the sensations and the resultant tic behaviour is strengthened. ER aims at interrupting the association, thus preventing the tics to occur. By confronting patients for a prolonged period of time with the sensations (exposure) and resisting the tic (response prevention), the patients might learn to tolerate the unpleasant sensation (habituation). Habituation will lessen the urge or need to give in to the tic, resulting in a reduction of tic behaviour. One RCT comparing 12 2-hour ER sessions (including 2 training sessions) with 10 1-hour HR sessions [START_REF] Verdellen | Exposure with response prevention versus habit reversal in Tourettes's syndrome: a controlled study[END_REF] revealed no differences between conditions (see Table 1). The study included both children and adults (age 7-55) with TS. Effect sizes were larger for ER than for HR. Results of this RCT and of a few case studies into ER [START_REF] Hoogduin | Exposure and response prevention in the treatment of Gilles de la Tourette's syndrome: Four case studies[END_REF] provide support for ER and indicate the need for further study. Also, review articles [START_REF] Cook | Evidence-based psychosocial treatments for tic disorders[END_REF][START_REF] Himle | Brief review of habit reversal training for Tourette syndrome[END_REF][START_REF] Peterson | Psychosocial management of tics and intentional repetitive behaviors associated with Tourette syndrome[END_REF][START_REF] Piacentini | Behavioral treatments for Tourette syndrome and tic disorders: state of the art[END_REF] suggest that ER is a promising treatment for tics. Preliminary results indicate that ER is effective for vocal and motor tics, for children as well as adults, for tic severity as well as tic frequency, with no indications of a rebound effect [START_REF] Verdellen | Tic suppression in the treatment of Tourette's syndrome with exposure therapy: the rebound phenomenon reconsidered[END_REF]. Since younger children are less aware of premonitory sensory motor phenomena it has to be clarified if there is an age effect respective an age limit for ER but also for HR [START_REF] Banaschewski | Premonitory sensory phenomena and suppressibility of tics in Tourette Syndrome -developmental aspects in children[END_REF].

Cognitive behavioural treatment (CBT)

Two studies (both included adults only) were identified that employed cognitive behavioural interventions (see table 1). The cognitive component of the intervention consisted of mentally challenging and restructuring the way patients evaluated their expectations and actions in high-risk situations related to frequent ticcing behaviour. One study compared CBT with HR in CTD and found comparable results for both treatments [START_REF] O'connor | A comparison of a behavioural and a cognitivebehavioural approach to the management of chronic tic disorders[END_REF]. In another study CBT combined with HR in CTD was found superior to WL [START_REF] O'connor | Evaluation of a cognitive-behavioural program for the management of chronic tic and habit disorders[END_REF]. The value of cognitive interventions in the treatment of tics remains to be investigated separately, as studies into CBT included a HR component, thus making its unique contribution to the results unclear.

Preliminary results seem to indicate that cognitive interventions have no specific additive

value in the treatment of tics.

Bio(neuro) feedback (NF)

NF is aimed at self-directed modulating of defined parts of brain electrical activity [START_REF] Heinrich | Annotation: neurofeedback -train your brain to train behaviour[END_REF]. It is based on operant conditioning; when brain activity changes in the desired direction, a positive 'reward' feedback is given to the individual.

Two case studies on TS (both N=2) were found, one with positive results [START_REF] Tansey | A simple and a complex tic (Gilles de la Tourette's syndrome): their response to EEG sensorimotor rhythm biofeedback training[END_REF], the other with inconclusive results [START_REF] Breteler | Psychologische interventies bij het syndroom van Gilles de la Tourette [Psychological interventions in Gilles de la Tourette syndrome[END_REF]. Also, a study in 15 TS patients showed significantly lower tics during relaxation biofeedback compared to arousal biofeedback [START_REF] Nagaia | Influence of sympathetic autonomic arousal on tics: Implications for a therapeutic behavioral intervention for Tourette syndrome[END_REF]. Considering the lack of randomized controlled research on NF and biofeedback for the treatment of tics, further research is required to allow for conclusions to be drawn.

Psychosocial interventions

The literature search and recent reviews by Cook and Blacher [START_REF] Cook | Evidence-based psychosocial treatments for tic disorders[END_REF] and Woods, Piacentini, & Walkup [START_REF] Woods | Treating Tourette syndrome and tic disorders: A guide for practitioners[END_REF] indicate there is no evidence-based practice with regard to supportive psychosocial interventions in TS. There is limited literature guiding interventions which modify the child's environment (parents, teachers, peers) to facilitate reduction of tics and enhance quality of life [START_REF] Conelea | The influence of contextual factors on tic expression in Tourette's syndrome: a review[END_REF][START_REF] Packer | Tic-related school problems: impact on functioning, accommodations, and interventions[END_REF]. However, education about tic disorders and the provision of support and reassurance to patients and their families have been described as the cornerstone for all other treatment interventions [START_REF] Peterson | The treatment of Tourette's syndrome: multimodal, developmental intervention[END_REF]. Therefore, based on clinical practice rather than empirical evidence, the present guideline includes recommendations on these approaches.

It is broadly accepted that TS and related tic disorders affect the lives of patients and all family members. Specifically, family members often have difficulty adjusting to the diagnosis and may struggle to manage with comorbid difficulties such as anger outbursts and hyperactivity [START_REF] Dooley | Parent perception of symptom severity in Tourette's syndrome[END_REF]. Psychoeducation represents an important component in providing support and resolving misunderstanding, uncertainty and stigma in TS [START_REF] Cutler | The quality of life of young people with Tourette syndrome[END_REF]. Information about the natural course of TS and helping the child and his environment to identify personal strengths often serves to reduce anxiety. It is proposed to provide relevant information to a child about the condition. This will promote coping strategies and self-efficacy and provide the child with the tools to explain to others (especially teachers and schoolmates) about the disorder.

Providing educators with general information relating to the aetiology, presentation, and course of TS will help them to implement effective individualised strategies to manage classroom behaviour, thereby allowing the child to maximize learning potential. Due to the rapidly changing nature of TS, Kepley & Conners [START_REF] Kepley | Management of learning and school difficulties in children with Tourette In[END_REF] highlight the need for a flexible approach, close liaison between parents and teachers and frequent re-evaluation of interventions within education to ensure effective management.

Children with TS report that they struggle to fit into society's expectations and rate associated embarrassment as disabling as the tics themselves [START_REF] Cutler | The quality of life of young people with Tourette syndrome[END_REF][START_REF] Wand | Tourette syndrome: associated symptoms and most disabling features[END_REF]. Young people with TS have been rated as less socially favourably as other non-affected peers in studies using laboratory-based paradigms [START_REF] Boudjouk | Negative peer evaluation in adolescents: Effects of tic disorders and trichotillomania[END_REF][START_REF] Friedrich | Children's attitudes and behavioral intentions toward a peer with Tourette syndrome[END_REF][START_REF] Woods | Evaluating the social acceptability of persons with habit disorders: The effects of topography, frequency, and gender manipulation[END_REF]. Two studies have looked at peer-perceptions of TS and the effects of psychoeducation through a video-tape intervention [START_REF] Friedrich | Children's attitudes and behavioral intentions toward a peer with Tourette syndrome[END_REF][START_REF] Woods | Controlled evaluation of an educational intervention used to modify peer attitudes and behavior toward persons with Tourette's Syndrome[END_REF]. In one study, older students viewed a TS-specific educational video. These students reported more positive attitudes toward people with tics than those who viewed other non-TS related material [START_REF] Woods | Controlled evaluation of an educational intervention used to modify peer attitudes and behavior toward persons with Tourette's Syndrome[END_REF]. Another study involving younger children showed no difference in attitudes following presentation of psychoeducational material. The effect on social behaviour intentions and actual social behaviour remains unclear and further research in this area is needed.

Group work

Case studies have been published describing and evaluating group work targeting different symptoms in children with TS. Group approaches offer children and their parents the opportunity to meet one another within a supportive environment. Groups can provide psychoeducational material [START_REF] Murphy | Group Work in Young People with Tourette Syndrome[END_REF] on aspects of TS such as tic management, anger management, bullying and school and self-esteem. Although the approach remains at the descriptive stage, qualitative feedback following such interventions has been positive and it seems worthy of further evaluation.

Charities and support organisations

The preponderance of charities for people with TS throughout the world are a vehicle for sharing information on TS. Taubert [START_REF] Taubert | Tourette's syndrome-tics obsessions compulsions: Developmental psychopathology and clinical care[END_REF] discusses the important role of voluntary organizations in supporting people with TS and their families. Large organisations such as the Tourette Syndrome Association in the US provide care and advocacy. Meanwhile, several European self-help organizations (e.g. in Germany, France, the Netherlands) are also quite active. While support groups for families are important, a more holistic approach between professionals, charities and government bodies may also be key role for such organisations.

Conclusions

The literature review provides scientific evidence for the behavioural treatment of tics in tic disorders. Most studies investigated the effectiveness of HR. Based on eight RCT's, several review articles and dozens of case studies, HR can be considered an evidence based behavioural treatment for tics for both children and adults. HR was found superior to WL [START_REF] Azrin | Treatment of Tourette Syndrome by habit reversal: A waiting-list control group comparison[END_REF][START_REF] O'connor | Evaluation of a cognitive-behavioural program for the management of chronic tic and habit disorders[END_REF], MP [START_REF] Azrin | Habit reversal vs negative practice treatment of nervous tics[END_REF], and SP [START_REF] Wilhelm | Habit reversal versus supportive psychotherapy for Tourette's disorder: a randomized controlled trial[END_REF][START_REF] Deckersbach | Habit reversal versus supportive psychotherapy in Tourette's disorder: a randomized controlled trial and predictors of treatment response[END_REF][START_REF] Piacentini | Behavior therapy for children with Tourette disorder: a randomized controlled trial[END_REF], and equally effective as ER [START_REF] Verdellen | Exposure with response prevention versus habit reversal in Tourettes's syndrome: a controlled study[END_REF]. A number of RCT's have shown that the improvement of HR continues up to at least ten months following completion of treatment. The literature also provides support for ER. In one RCT [START_REF] Verdellen | Exposure with response prevention versus habit reversal in Tourettes's syndrome: a controlled study[END_REF] comparing ER with HR, larger effect sizes were found for ER than for HR, indicating its efficacy. In addition, several review articles and a few case studies demonstrate that ER is a promising, probably efficacious treatment for tic disorders for both children and adults, which needs further research.

Although most evidence was found for HR and, to a lesser extent, for ER, the current literature base is limited by the availability of only one (albeit large-scale) RCT [START_REF] Piacentini | Behavior therapy for children with Tourette disorder: a randomized controlled trial[END_REF] with a child and adolescent-only sample. Therefore, firm conclusions for these specific age groups cannot be drawn. Comparability of the RCT's was limited by the different outcome measures that were used, application of different HR ingredients, different sample ages, number of sessions, and follow-up periods. Only three RCT's [START_REF] Wilhelm | Habit reversal versus supportive psychotherapy for Tourette's disorder: a randomized controlled trial[END_REF][START_REF] Piacentini | Behavior therapy for children with Tourette disorder: a randomized controlled trial[END_REF][START_REF] Verdellen | Exposure with response prevention versus habit reversal in Tourettes's syndrome: a controlled study[END_REF] reported effect sizes. In order to enable comparisons of efficacy, future studies are encouraged to include such calculations.

There are no controlled studies into the efficacy and effectiveness of other psychosocial interventions, e.g., psychoeducation or support groups. Therefore, these interventions are not to be considered fully evidence based, although the importance of these interventions is emphasized in clinical practice.

Recommendations

Based on the evidence at present and clinical support this guideline recommends both HR and ER as first line behavioural treatments for tics for both children and adults. HR has a broader evidence base and is usually offered. Results indicate that ER may be more effective than HR at the severe end of TS, i.e., when many tics are involved [START_REF] Verdellen | Exposure with response prevention versus habit reversal in Tourettes's syndrome: a controlled study[END_REF]. However, ER as applicated in the RCT took twice as much therapy time as HR, i.e., studies were based on sessions lasting 2 hours each, whereas HR sessions usually last 1 hour. Recent research shows that ER sessions lasting for 1 hour had comparable results [START_REF] Jvd | ) The effect of exposure length on treatment outcome for tics in Tourette's syndrome: One hour sessions versus two hour sessions[END_REF]. Other treatments for tics were identified in the literature, for which insufficient evidence was found. MP, the oldest behavioural treatment for tics reported in the literature, lacks evidence and therefore is not recommended as a treatment for tics. CM/reinforcement techniques and FBI are also not to be considered evidence based treatments for tics. However, case studies indicate that these interventions have some value in specific cases and need further research. CM may be a useful tool for children, because motivation for a behavioural treatment may not be intrinsic and its reward (i.e., tic reductions) may be too far away for a child. Based on the current literature, CM and FBI are recommended as second line behavioural interventions. RT, although also not to be considered an evidence based treatment for tics, is recommended as a second line behavioural treatment for tics as well. Case studies indicate that patients can benefit from this treatment to some extent and hypnosis and cue-controlled relaxation were found to reduce tics merely via stress reduction in specific cases. Cognitive interventions, although these have not been studied in adolescents, do not seem to have additive value to HR. Therefore, at present, this method is not recommended as an intervention for tics. Based on the sparse and inconclusive literature on the efficacy of NF, this method cannot yet be recommended as a treatment for tics. However, a RCT in children with ADHD showed a long-term improvement on selfregulation of behaviour, which seems promising also for TS, especially in cases where TS and ADHD may coexist [START_REF] Gevensleben | Is neurofeedback an efficacious treatment for ADHD? A randomised controlled clinical trial[END_REF][START_REF] Gevensleben | Neurofeedback training in children with ADHD: 6-month follow-up of a randomised controlled trial[END_REF]. NF should thus be further evaluated as a treatment for tics.

Although there are no controlled studies into the efficacy and effectiveness of other psychosocial interventions, e.g., psychoeducation, bibliotherapy, self help, social support groups, this guideline recommends embedding each treatment within a psychoeducational and supportive context. Psychoeducation alone may also be useful for families who do not engage with more comprehensive treatments due to very mild tics or lack of services. Specific interventions must target not only tic symptoms, but also comorbid problems and coping strategies that can profoundly influence the impact that TS may have on an individual's well being during childhood and later into adulthood. The present guideline does not provide recommendations as to the behavioural treatment of comorbidity in tic disorders.

-------------Include Figure 1 here --------------A summary of the recommendations of this guideline is presented in a flowchart (see Figure 1). Psychoeducation is recommended in all (both mild and more severe) cases. Other supportive interventions (e.g., liaison with school, group work, social skills training) are recommended in individual cases depending on the specific needs of a child and his environment. HR and ER are behavioural treatments recommended as first line treatments to patients who want to reduce their tics. In helping a child or adolescent (and his family) making a choice which treatment to start with, information should be given about both treatments. This guideline recommends to evaluate treatment response after 10 sessions, and to continue treatment for another 10 sessions when there is a partial response, to start the alternative treatment when there is no response, and to consider medication when both treatments have not resulted in relevant tic reductions. In case of severe symptoms, it is recommended to consider combining a behavioural treatment with medication or starting

  medication first and adding behavioural treatment later. Based on research at present, weekly sessions of 1 hour are advised in HR and weekly sessions of 2 hours in ER. In addition, second line interventions such as CM, FBI or RT are recommended in individual cases, when first line interventions do not suffice.

Figure 1

 1 Figure 1Decision tree of psychological interventions for tics in tic disorders

Table 1

 1 Randomized controlled trials of behavioural interventions for tics disorders (divided according to the inclusion or exclusion of children)

				Tic Disorder
		Mild symptoms	Moderate symptoms	Severe symptoms
	Monitoring
				Psychoeducation
				Psychosocial impairment		Medication (see corresponding
				Preference of child / parents		guideline this issue)
				Availability of intervention
		Habit reversal			Exposure and
					Response Prevention
	Continue	Satisfactory response after 10 sessions?		Switch	Satisfactory response after 10 sessions?	Continue
	Partly	Yes	No	No	Yes	Partly
		Stop			Stop
		Both interventions unsatisfactory: consider medication/
				combination with medication

Tic frequency home (report of parent/partner):

  CTD=chronic tic disorder; MP=massed practice; WL=waiting list; HR=habit reversal; ER=exposure and response prevention; RT=relaxation training; MT=minimal therapy; SP=supportive psychotherapy; CBT=cognitive behavioural therapy; YGTSS=Yale Global Tic Severity Scale; * = significant difference p<0.05; ** = significant difference p<0.01; ES=effect size; FU=follow-up

	average 11,7) 32 TS patients (average age 36,2) Children & Adults HR, SP parallel Children only Adults only average 39,1) TS=Tourette Syndrome; (age 18-60, Wilhelm et al., 2003	HR>SP*	YGTSS: HR: 30.5 pre-19.8 post (ES 1.50) SP: 26.6 pre-26.9 post (ES -0.03) frequency and intensity on diary and video measures WL: no significant decrease Degree of control: HR/CBT: 65% of patients reported between 75 and 100% control	SP: 9.9 (n=6) 6 months: YGTSS: HR: 13.3 (n=23) SP: 10.4 (n=6) 10 months: YGTSS: HR: 21.0 (still significantly improved) SP: 23.8 (pre-treatment level) degree of control: HR/CBT: maintained results 2 years: Degree of control: HR/CBT: 52% of patients maintained 75-100% control	HR consisted of awareness training, competing respons training, relaxation training and function based intervent SP consisted of supportive psychotherapy and educatio Significantly more children receiving HR were rated as very much improved or much improved on the Clinical G Impression-improvement scale (52.5% vs 18.5%) 3 and 6 months follow-up: results remained stable for th available responders 14 sessions (8 weekly, 6 2-weekly) HR consisted of self-monitoring, competing response tr relaxation training and contingency management HR improved significantly on the Clinical Global Impres improvement scale (CGI-i) as well, SP not Both tic disorders (n=47) and habit disorders (n=43) we included HR consisted of awareness training, competing respons training, relaxation training, relapse prevention and was accompanied with more general cognitive and behaviou restructuring 2 years FU: interview by telephone
							10 months FU: no significant difference between HR an
							the YGTSS; significant difference between HR and SP
							maintained on the CGI-i
	Deckersbach et al., 2006 O'Connor et al., 1997	HR, SP paralell HR, CBT Parallel	30 TS patients (average age 35,1) 14 CTD patients (age 23-49)	HR>SP* HR=CBT	YGTSS: HR: 39.3 pre-18.3 post SP: 27.7 pre-26.8 post Tic frequency (diary): HR: 54% decrease CBT: 57% decrease Degree of control:	3 months: YGTSS: ER: 14.0 (ES 1.49) HR: 13.5 (ES 1.95) 6 months: YGTSS: HR: 18.4 SP: 26.6 3 months: Tic frequency (diary): HR: 77% decrease CBT: 86% decrease	14 sessions (8 weekly, 4 2-weekly, 2 monthly) HR consisted of awareness training, self-monitoring, co response training, relaxation training and contingency 12 sessions ER (2 hours, weekly) vs 10 sessions HR (1 management weekly) Both groups improved in life-satisfaction and psychosoc Weighted gain scores were used to correct for number functioning; only HR improved significantly in tic severit HR consisted of awareness training and competing resp 6 months FU: results remained stable training HR and ER improved significantly on all measurements FU included 25 patients who had received the alternativ 10 sessions (weekly) HR consisted of awareness training, competing respons and contingency management condition CBT included HR elements; cognitive approach showed
	Bergin et al., 1997 Piacentini et al., 2010	RT, MT parallel HR, SP parallel	23 TS patients (age 7-18, average 11,3) 126 TS/CTD patients (age 9-17, patients	RT=MT HR>SP	HR: 65% ER: 89% of patients showed >30% reduction (ES 0.88) HR: 72% of patients showed >30% reduction (ES 0.73) YGTSS: RT: 86% of patients improved (non-significant) MT: 66% of patients improved (non-significant) CBT: 78% YGTSS: HR: 24.7 pre-17,1 post (ES 0.68) SP: 24.6 pre-21,1 post	Degree of control: 6 weeks: YGTSS: RT: 71% of patients improved (non-significant) MT: 55% of patients improved HR: 69% CBT: 87% 2 years: Degree of control: HR: 43% of patients had maintained degree of control CBT: 50% of patients had (non-significant) maintained degree of control 3 months: 2 months: YGTSS: HR:13.9 (n=23) Tic frequency, intensity and	significant additional effect 2 years FU: interview by telephone 6 sessions (1 hour, weekly) No significant improvements Comparable results on other tic severity measurements Videotape Scale, Hopkins Motor and Vocal Tic Scale, T Syndrome Severity Scale) Group non-RCT design 12 sessions (weekly)

HR/CBT>WL* Tic frequency and intensity (diary, video):

HR/CBT: 88% of patients significant decrease in

sessions (during 10 weeks; first two sessions 1,5 hou remaining sessions 1 hour)


MD Francesco Cacciatore
email: francesco.cacciatore@fsm.it

MD b Giuseppe Bruzzese

MD a , Dino Franco Vitale

MD b Antonio Liguori

Filomena Nigris

MB c , Carmela Fiorito

BiolD Teresa Infante

MD Francesco Donatelli

Biagio Pellegrino

Louis Joseph Minucci

Ignarro

BiolD, PhD c , Filomena De Nigris

BiolD Minucci

PhD Louis Joseph Ignarro

MD Claudio Napoli

Effects of ACE inhibition on circulating endothelial progenitor cells, vascular damage, and oxidative stress in hypertensive patients

Keywords: ACE inhibitors, EPC, vascular damage, hypertension

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

Introduction

In the last decade, there was a growing interest in the regenerative medicine afforded by the findings related to endothelial progenitor cells (EPCs) from bone marrow origin. [START_REF] Napoli | Therapeutic approaches in vascular repair induced by adult bone marrow cells and circulating progenitor endothelial cells[END_REF][START_REF] Napoli | Novel pathogenic insights in the primary prevention of cardiovascular disease[END_REF] Thus, the pathobiology of EPC is an emerging field in the clinical setting of cardiovascular diseases. It has been hypothesized that EPC depression can be causally linked to the atherogenic process. [START_REF] Fadini | Peripheral blood CD34 + KDR + endothelial progenitor cells are determinants of subclinical atherosclerosis in a middle-aged general population[END_REF] EPCs constitute a circulating pool of cells able to repair the dysfunctional endothelium whereas a reduced EPC pool may determine the inability to maintain adequate endothelial homeostasis. Moreover, the number and the functional activity of EPCs have been reported to decrease with hypertension, diabetes mellitus and the coexistence of multiple classical cardiovascular risk factors. [START_REF] Heitzer | Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease[END_REF][START_REF] Vasa | Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease[END_REF][START_REF] Liguori | Functional impairment of hematopoietic progenitor cells in patients with coronary heart disease[END_REF][START_REF] Kinnaird | Cardiovascular risk factors impair native collateral development and may impair efficacy of therapeutic interventions[END_REF][START_REF] Spinetti | Diabetes and vessel wall remodelling: from mechanistic insights to regenerative therapies[END_REF][START_REF] Feihl | Hypertension and microvascular remodelling[END_REF][START_REF] Lijnen | Angiogenesis and obesity[END_REF][START_REF] Balestrieri | High glucose downregulates endothelial progenitor cell number via SIRT1[END_REF]. Levels of EPCs are also inversely correlated to progression of coronary heart disease. [START_REF] Briguori | Correlations between progression of coronary artery disease and circulating endothelial progenitor cells[END_REF] There are some preclinical studies suggesting that ACE inhibition may have potential effects on EPC. [START_REF] You | Combination of the Angiotensin-Converting Enzyme inhibitor Perindropil and diuretic indapamide activate postnatal vasculogenesis in spontaneously hypertensive rats[END_REF][START_REF] Zambidis | Expression of angiotensine-converting enzyme (CD 143) identifies and regulate primitive hemangioblasts derived from human pluripotent stem cells[END_REF][START_REF] Muller | ACE inhibition promotes upregulation of endothelial progenitor cells and neoangiogenesis in cardiac pressure overload[END_REF] Since ACE inhibitors are widely used in cardiovascular therapy, this promoted further investigation into the effects of such compounds on circulating EPC and their role in vascular healing, repair, and post natal neovascularization. ACE inhibitors may have a pivotal role in the management of atherosclerotic-related diseases independent of their vasodilating and hypotensive effects.

Experimental studies show that these drugs can attenuate the development of atherosclerosis in a wide range of species, the most effective being those containing the antioxidant sulfhydryl group. [START_REF] Napoli | Beneficial effects of ACE-inhibition with zofenopril on plaque formation and low-density lipoprotein oxidation in Watanabe heritable hyperlipidemic rabbits[END_REF][START_REF] Chobanian | Antiatherogenic effect of captopril in the Watanabe heritable hyperlipidemic rabbit[END_REF][START_REF] Hayek | Antiatherosclerotic and antioxidative effects of captopril in apolipoprotein E-deficient mice[END_REF][START_REF] Sun | Comparative effects of ACE inhibitors and an angiotensin receptor blocker on atherosclerosis and vascular function[END_REF][START_REF] Candido | Prevention of accelerated atherosclerosis by angiotensin-converting enzyme inhibition in diabetic apolipoprotein Edeficient mice[END_REF] Here, we used a previous clinical trial [START_REF] Napoli | Long-term treatment with sulfhydryl angiotensin-converting enzyme inhibition reduces carotid intima-media thickening and improbe the nitric oxide/oxidative stress pathways in newly diagnosed patients with mild to moderate primary hypertension[END_REF] on ACE inhibition and vascular damage to study EPC number and function and the relation to carotid intimal medial thickness (IMT) progression over the time.

Methods

Participants and measurements

We studied 36 newly diagnosed mildly hypertensive patients; this population comes from a previously published ongoing study [START_REF] Napoli | Long-term treatment with sulfhydryl angiotensin-converting enzyme inhibition reduces carotid intima-media thickening and improbe the nitric oxide/oxidative stress pathways in newly diagnosed patients with mild to moderate primary hypertension[END_REF] involving 48 patients and started in 2001. Exclusion criteria included the presence of other classical risk factors for coronary heart disease (eg, diabetes/impaired glucose tolerance and hyperlipidemia); prior or concurrent therapy with ACE inhibitors, antiplatelet drugs, or anticoagulants; history of ischemic events; or refusal to grant informed consent. Patients were enrolled if they had systolic BP >160 mmHg and/or diastolic BP >95 mmHg. Hyperlipidemia was defined as having total cholesterol >220 mg/dL, or LDL cholesterol >140 mg/dL, or triglyceride >150 mg/dL, and/or taking antihyperlipidemic medications.

We used a simple randomization scheme with treatment assignment determined by computerized random number generation in a sequential manner and then provided to the clinical investigators in sealed envelopes. Follow-up measurements were scheduled at 1 and 5 years. The trial protocol followed the principles outlined in the Declaration of Helsinki and approved by the Local Ethical Committee.

Carotid ultrasonography measurements

All carotid ultrasonography measurements were made by 2 investigators (G.B. and A.L.) to eliminate interobserver variability, blinded to treatment randomization, as previously described.

[21] Carotid artery IMT was measured by high-resolution B-mode ultrasonography, with a 7.5-MHz high-resolution transducer (Sonos-5500 system; Philips Medical Systems, Andover, MA) as described previously. [START_REF] Napoli | Rethinking primary prevention of atherosclerosis-related diseases[END_REF][START_REF] Napoli | Relations between vasoactive hormones and diastolic function in hypertensive uraemic patients[END_REF] The carotid arteries were carefully examined for wall changes in the longitudinal and transverse views. The common carotid artery, carotid bulb, and internal and external arteries were examined. Presence of carotid atherosclerotic lesions (plaques or shadowing) was determined from scans of all right and left carotid artery segments (common carotid artery, bifurcation, and internal carotid artery). The presence of plaques was defined during ultrasound reading based on wall thickness and arterial wall roughness, loss of alignment, or protrusion into the lumen. Calcification or mineralization, another indicator of atherosclerosis, was based on acoustic shadowing. Right and left common carotid artery wall areas were calculated as the total artery area minus the lumen area assuming a circular lumen and an outer artery structure that was either circular or elliptical. The IMT of the far wall was defined as the distance from the lumen-intima interface (leading edge of the first echogenic line) to the media-adventitia interface (leading edge of the second echogenic line). We measured the IMT of the common carotid arteries bilaterally at 3 points (at 1, 2, and 3 cm proximal to the carotid bifurcation); the averaged value was taken as the carotid IMT. IMT values for the 3 different projections were averaged to obtain the mean maximum

IMT (MM-IMT).

Lesions of apparent plaque appearing either as faint gray echoes (soft plaques) or bright white echoes (calcified plaque) protruding into the lumen, were always excluded from the IMT measurement. [START_REF] Persson | Noninvasive quantification of atherosclerotic lesions. Reproducibility of ultrasonographic measurement of arterial wall thickness and plaque size[END_REF] Blood pressure measurements Blood pressure (BP) was measured at baseline and during the 2 subsequent examinations with a mercury sphygmomanometer in a standardized fashion, after 10 minutes of rest with the subject in the supine position. [START_REF] Napoli | Sulfhydryl angiotensin-converting enzyme inhibition induces sustained reduction of systemic oxidative stress and improves the nitric oxide pathway in patients with essential hypertension[END_REF] Systolic and diastolic BPs (mmHg) were defined according to Korotkoff sounds I and V. All BP measurements were done just before carotid ultrasound measurements and were performed after drug administration by a physician who was blinded as to the patients' profiles and treatment assignments.

Evaluation of plasma nitrite/nitrate and isoprostane concentrations

Plasma nitrite/nitrate (NOx) levels were measured with the classical Griess method, as previously described. [START_REF] Napoli | Sulfhydryl angiotensin-converting enzyme inhibition induces sustained reduction of systemic oxidative stress and improves the nitric oxide pathway in patients with essential hypertension[END_REF] Amounts of plasma nitrite were estimated by a standard curve obtained from enzymatic conversion of NaNO3 to nitrite. Isoprostanes (8-iso-PGF2α) were measured from plasma samples by using a commercially available immunoassay (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. [START_REF] Napoli | Sulfhydryl angiotensin-converting enzyme inhibition induces sustained reduction of systemic oxidative stress and improves the nitric oxide pathway in patients with essential hypertension[END_REF] Measurement of EPC number (culture assay) Mononuclear cells were isolated by density gradient centrifugation with Biocoll (Biochrom, Berlin, Germany) from 20 mL peripheral blood, as previously described. [START_REF] Casamassimi | Comparison between total endothelial progenitor cell isolation versus enriched Cd133+ culture[END_REF] After isolation, 4×10 6 mononuclear cells were plated on 24-well culture dishes coated with human ibronectin (Sigma-Aldrich, Munich, Germany) and maintained in endothelial basal medium (Cambrex, Walkerville, MD, USA) supplemented with endothelial growth medium and 20% fetal calf serum. After 4 d in culture, non-adherent cells were removed by thorough washing with phosphate-buffered saline (PBS).

Measurement of functional capacity of EPCs (migrating capacity)

Isolated EPCs were detached using 1 mmol/L ethylenediaminetetraacetic acid in PBS (pH 7.4), harvested by centrifugation, resuspended in 500 µL endothelial basal medium, counted, and placed in the upper chamber of a modified Boyden chamber (2 × 104 cells; BD Bioscience, Milan, Italy).

The chamber was placed in a 24-well culture dish containing endothelial basal medium, 20% fetal calf serum, and human recombinant vascular endothelial growth factor (VEGF) (50 ng/mL; R&D Systems, Minneapolis, MN, USA). After 24 h incubation at 37°C, the lower side of the filter was washed with PBS and fixed with 2% paraformaldehyde. For quantification, cell nuclei were stained with 4',6'-diamidino-2-phenylindole. Cells migrating into the lower chamber were counted manually in several random microscopic fields. [START_REF] Casamassimi | Comparison between total endothelial progenitor cell isolation versus enriched Cd133+ culture[END_REF] Measurements were blinded to treatment randomization

Statistical analysis

Data are presented as mean ± SD. ANOVA for repeated measure was used to test the effect of treatment (enalapril vs zofenopril) over the time (Baseline, 1 year and five years) on systolic and diastolic BP, IMT, EPCs, Migratory cells, NOx and PGE 2alpha , in the table are reported statistical significance for the treatment effect, time effect and interaction between treatment and time. In order to assess the association between IMT versus EPC and/or Migratory cells controlling for confounder effect of treatment, time and other variables, we used linear regression analysis with robust variance estimation clustering for each patients in order to compensate for the correlation between the repeated measures. [START_REF] Williams | A note on robust variance estimation for cluster-correlated data[END_REF] A p value < 0.05 was considered as statistically significant.

Analysis was performed using STATA 10 software.

Results

The mean age of the patients was 41.6±2.6 (range [START_REF] Evangelista | Antioxidant and cardioprotective properties of the sulphydryl angiotensin-converting enzyme inhibitor zofenopril[END_REF][START_REF] Cominacini | Zofenopril inhibits the expression of adhesion molecules on endothelial cells by reducing reactive oxygen species[END_REF][38][39][40][41][42][43][44][45][46]. No significant difference in age, gender, family history of an ischemic event (CHD, peripheral arterial disease, or stroke), history of smoking, sedentary lifestyle, or medication used (calcium antagonist; 2 in enalapril and 2 in zofenopril group) were present at baseline between the 2 groups [START_REF] Napoli | Long-term treatment with sulfhydryl angiotensin-converting enzyme inhibition reduces carotid intima-media thickening and improbe the nitric oxide/oxidative stress pathways in newly diagnosed patients with mild to moderate primary hypertension[END_REF]. Moreover, no differences were present for others risk factors such as total cholesterol, triglycerides, high-density lipoprotein cholesterol, or LDL-cholesterol. [START_REF] Napoli | Long-term treatment with sulfhydryl angiotensin-converting enzyme inhibition reduces carotid intima-media thickening and improbe the nitric oxide/oxidative stress pathways in newly diagnosed patients with mild to moderate primary hypertension[END_REF] No patients had diabetes or were in treatment with statin or aspirin. The compliance to pharmacological treatment was good without withdrawn during the follow-up.

EPCs increased during the follow-up with no statistical differences between treatment groups.

(Table 1 and Figure 1). Migrating capacity of EPCs did not change during the study and no differences were evident between the treatment groups. (Table 1 and Figure 1). Plasma nitrite/nitrate (NOx) decreased significantly during the study while no differences were evident between the treatment groups. (Table 1 and Figure 1). Isoprostanes (8-iso-PGF2α) decreased significantly during the study more markedly in zofenopril treated patients. (Table 1 and Figure 1).

Intimal media thickness increased during the follow-up and this effect was more evident in the group of patients treated with enalapril even if this difference did not reach statistical significance when compared to zofenopril treated group. A statistically significant interaction between treatments and time suggesting a time related effect of pharmacological treatment in reducing IMT progression. (Table 1 and Figure 1). As expected, a significant reduction of systolic and diastolic blood pressure during the study was obtained with no statistical differences between treatment groups also considering the effect of interaction between treatment and time. (Table 1 and Figure 1).

In order to assess the correlation between IMT and EPCs and migratory cells, adjusted for all other covariates, we built a multiple linear regression model with IMT as independent variable and EPC and migratory cells as effect modifiers and all the remaining variables listed in the table as confounder. The multiple linear regression model showed that the IMT was significantly inversely correlated with EPC but not with migratory cells after adjustment for confounders. (Table 2 and Figure 2)

Discussion

The major findings of our study are: 1) an EPC increase during the follow-up in both groups of patients treated with enalapril and zofenopril, 2) an inverse correlation between circulating EPCs and IMT increase over the time, 3) a plasma nitrite/nitrate decrease without evident differences between the treatment groups and 4) a isoprostanes decrease more evidently in zofenopril treated patients.

Migrating capacity of EPCs did not change during the study and no differences were evident between the treatment groups. Multivariate analysis demonstrated that vascular damage was significantly inversely correlated with EPCs levels but not with migrating capacity after adjustment for confounders.

A previous cross-sectional study, demonstrated an association of EPC migration, age and mean arterial blood pressure on carotid intimal thickning, they also found an inverse correlation with EPC number (r = -0.349) but with no statistical significance (p=0.063) demonstrating an age related decrease in EPC function and a possible effect on vascular remodelling. [START_REF] Keymel | Impaired endothelial progenitor cell function predicts age-dependent carotid intimal thickening[END_REF] Another study [START_REF] Lau | Burden of carotid atherosclerosis in patients with stroke: relationships with circulating endothelial progenitor cells and hypertension[END_REF] demonstrated that a significantly higher carotid mean maximum intima-media thickness, prevalence of carotid plaque and a lower number of circulating CD34 + /KDR + EPCs was found in stroke patients, compared with controls. In stroke patients, circulating CD34 + /KDR + EPCs count correlated negatively with carotid IMT, and was an independent risk factor for increased carotid IMT > 1 mm and the presence of carotid plaque.

Another very recent study [START_REF] Porto | Comparison of the effects of ramipril versus telmisartan on high-sensitivity C-reactive protein and endothelial progenitor cells after acute coronary syndrome[END_REF] established that telmisartan shows a more potent anti-inflammatory effects than the ACE inhibitor ramipril and the two drugs did not show a differential effects on EPCs.

Since the expression of ACE may regulate primitive hemangioblasts derived from human stem cells, [START_REF] Zambidis | Expression of angiotensine-converting enzyme (CD 143) identifies and regulate primitive hemangioblasts derived from human pluripotent stem cells[END_REF] the renin-angiotensin-aldosterone axis can be therefore exploited to direct the EPC fate during vascular regeneration. This issue may constitute an important clinical implication of ACE therapy on vascular damage occurring in hypertensive patients. ACE inhibitors may have a pivotal role in the management of atherosclerotic-related diseases independent of their vasodilating and hypotensive effects. Experimental studies showed that these drugs can attenuate the development of atherosclerosis in a wide range of species, the most effective being those containing the antioxidant sulfhydryl group. [START_REF] Napoli | Beneficial effects of ACE-inhibition with zofenopril on plaque formation and low-density lipoprotein oxidation in Watanabe heritable hyperlipidemic rabbits[END_REF][START_REF] Chobanian | Antiatherogenic effect of captopril in the Watanabe heritable hyperlipidemic rabbit[END_REF][START_REF] Hayek | Antiatherosclerotic and antioxidative effects of captopril in apolipoprotein E-deficient mice[END_REF][START_REF] Sun | Comparative effects of ACE inhibitors and an angiotensin receptor blocker on atherosclerosis and vascular function[END_REF][START_REF] Candido | Prevention of accelerated atherosclerosis by angiotensin-converting enzyme inhibition in diabetic apolipoprotein Edeficient mice[END_REF] Different ACE inhibitors have quite different chemical functional groups, and these structural variations may account for different in vivo and in vitro effects. The ACE inhibitor captopril has a sulfhydryl group to coordinate the zinc ion of the active site, enalaprilat has a carboxylate group, and zofenopril has 2 sulfhydryl groups. [START_REF] Kowala | Inhibitors of angiotensin converting enzyme decrease early atherosclerosis in hyperlipidemic hamsters. Fosinopril reduces plasma cholesterol and captopril inhibits macrophage-foam cell accumulation independently of blood pressure and plasma lipids[END_REF][START_REF] Unger | The role of the renin-angiotensin system in the development of cardiovascular disease[END_REF] Sulfhydryl ACE inhibition stimulates the NO activity and decreases oxidative stress in human endothelial cells [START_REF] Jacoby | Renin-angiotensin system and atherothrombotic disease: from genes to treatment[END_REF] and in patients with essential hypertension. [START_REF] Napoli | Long-term treatment with sulfhydryl angiotensin-converting enzyme inhibition reduces carotid intima-media thickening and improbe the nitric oxide/oxidative stress pathways in newly diagnosed patients with mild to moderate primary hypertension[END_REF] Zofenopril increases NO production in endothelium, decreases atherosclerotic development, and reduces reactive oxygen species, [START_REF] Scribner | The effect of angiotensin converting enzyme inhibition on endothelial function and oxidant stress[END_REF][START_REF] De Nigris | Chronic treatment with sulfhydryl angiotensin-converting enzyme inhibitors reduce susceptibility of plasma LDL to in vitro oxidation, formation of oxidation-specific epitopes in the arterial wall, and atherogenesis in apolipoprotein E knockout mice[END_REF][START_REF] Evangelista | Antioxidant and cardioprotective properties of the sulphydryl angiotensin-converting enzyme inhibitor zofenopril[END_REF][START_REF] Cominacini | Zofenopril inhibits the expression of adhesion molecules on endothelial cells by reducing reactive oxygen species[END_REF] as well as susceptibility to oxidation of plasma lowdensity lipoproteins (LDL) and formation of oxidation-specific epitopes in the arterial wall and atherogenesis in apolipoprotein E knockout mice. [START_REF] De Nigris | Chronic treatment with sulfhydryl angiotensin-converting enzyme inhibitors reduce susceptibility of plasma LDL to in vitro oxidation, formation of oxidation-specific epitopes in the arterial wall, and atherogenesis in apolipoprotein E knockout mice[END_REF] The meaning of NOx levels would be qualitatively rather than quantitatively. However, repeated assays in same subjects have high sensitivity. In the present study, as expected, the untreated state of hypertension was associated to increased levels of NOx, after both treatments, NOx level decreased indicating that a normalization of blood pressure was associated to restoring effects of the NO pathway.

Circulating EPCs could play an important role in accelerating endothelialization at areas of vascular damage, and EPC function would be a viable strategy for assessing reparative ability.

Consistently, some studies established that EPCs are affected both in number and function by several cardiovascular risk factors as well as various cardiovascular disorders, such as hypertension, hypercholesterolemia, and coronary heart disease [38].

Our study had some limitations. First, we used plasma storage collected from another clinical trial. [START_REF] Napoli | Long-term treatment with sulfhydryl angiotensin-converting enzyme inhibition reduces carotid intima-media thickening and improbe the nitric oxide/oxidative stress pathways in newly diagnosed patients with mild to moderate primary hypertension[END_REF] Another study limitation would be the limited power of the study due to a small group of patients.

In conclusion, our study demonstrated that EPC levels increased during the treatment in both groups of patients treated with ACE-inhibitors indicating that these drugs prevent vascular damage in newly hypertensive treated patients not only reducing blood pressure but also acting on circulating EPCs.

Author Contributions

ld)

Figure 1 .Figure 2 :

 12 Figure 1. Modifications during the study of parameters in enalapril-treated patients (circle &

Table 1 :

 1 Repeated Measure of Analysis of Variance in Enalapril (18-patients) and Zofenopril (18patients) groups during the study.

	Variable	Baseline	1 year	5 year	Treatment	Time	Interaction Treatment*time
	SBP -Enalapril SBP -Zofenopril	169.1±7.0 168.6±7.9	134.3±6.7 139.6±8.3	131.8±7.4 134.5±6.8	P=0.092	P=0.001	P=0.245
	DBP-Enalapril DBP -Zofenopril	101.9±5.8 102.6±8.3	81.3±5.6 76.0±3.5	75.9±6.6 76.2±6.1	P=0.199	P=0.001	P=0.050
	IMT -Enalapril IMT -Zofenopril	0.71±0.05 0.72±0.04	0.74±0.05 0.73±0.05	0.83±0.09 0.76±0.07	P=0.074	P=0.001	P=0.017

Table 2 :

 2 Multivariate linear regression analysis with robust variance estimation clustering for each patient in order to compensate for the correlation between repeated measures and IMT as dependent variable.

	Variable	B coefficient	Robust SE	P
	EPC #	-0.0018	0.0005	0.001
	Treatment #	0.0198	0.0152	0.203
	Time #	0.0861	0.0181	0.001
	Treatment * Time #	-0.0699	0.02	0.001
	EPC Migration	0.0054	0.0065	0.41
	NOx	-0.0005	0.0012	0.678
	8-iso-PGF2α	-0.0024	0.0011	0.04
	Systolic BP	0.0013	0.0008	0.1
	Diastolic BP	0.00005	0.0008	0.95
	Age	0.0013	0.0018	0.46
	Gender	0.0019	0.012	0.87

# Variable retained after backward hierarchical model.

Acknowledgments

. This work was supported in part by grants from the Progetto di Rilevante Interesse Nazionale Ministero Italiano Università e Ricerca 2006 [Code 0622153_002 "Meccanismi fisiopatologici di danno vascolare/trombotico ed angiogenesi" (to C.N.), Ricerca Finalizzata del Ministero della Salute 2007 (to F.C.) and the Fondation Jerome Lejeune, France (to C.N.).

Conflict of