Feasibility , reliability , and validity of adolescent health status measurement by the Child Health Questionnaire Child Form -LRB- CHQ-CF -RRB- : internet administration compared with the standard paper version Aims In this study we evaluated indicators of the feasibility , reliability , and validity of the Child Health Questionnaire-Child Form -LRB- CHQ-CF -RRB- . We compared the results in a subgroup of adolescents who completed the standard paper version of the CHQ-CF with the results in another subgroup of adolescents who completed an internet version , i.e. , an online , web-based CHQ-CF questionnaire . Introduction During the past two decades , several measures have become available to describe generic health-related quality of life in pediatrics , but adolescent self-report questionnaires received relatively little attention until now -LSB- 1 , 2 -RSB- . The Child Health Questionnaire -LRB- CHQ -RRB- is one of the most widely used pediatric health-related quality of life measures and has been translated into 21 languages -LRB- 32 countries -RRB- . There is a form for parents and also a self-report form for adolescents , the Child Health Questionnaire-Child Form -LRB- CHQ-CF -RRB- -LSB- 2 -- 7 -RSB- . The CHQ covers physical and psychosocial aspects of health , and includes the impact of child health problems or handicaps on family life -LSB- 3 -RSB- . This study focuses on the evaluation of missing answers at the item level , distribution of the scale scores , reliability , and validity of the CHQ-CF in an adolescent population . We expect the commonly used paper format of the CHQ and other health questionnaires to be increasingly replaced by internet versions , especially in adolescent populations that are accustomed to the use of computers and the internet -LSB- 8 -RSB- . From the perspective of clinicians and researchers , the advantages of using the internet include avoiding paper work , on-line data-entry , and procedures designed to reduce the number of missing answers and the length of questionnaires -LSB- 9 , 10 -RSB- . In general , the mode of questionnaire administration -LRB- e.g. , written questionnaire , face to face interview , telephone interview , computer questionnaire -RRB- may affect the participation rate , number of missing answers , psychometric properties , and actual scores -LSB- 11 -- 14 -RSB- . With regard to health questionnaires , several studies demonstrated some differences between the commonly used paper versions and computer versions of the same questionnaires -LSB- 15 -- 17 -RSB- . Especially in studies comparing paper and computer questionnaires on sensitive topics , administration via computer was found to increase reporting of e.g. , drug use or unsafe sexual behaviors , as this medium is apparently perceived as providing more privacy than a paper form -LSB- 18 -- 20 -RSB- . With regard to online , i.e. , internet or web-based administration of health questionnaires , several studies have demonstrated that online health questionnaires are feasible in various settings , especially among adolescents -LSB- 21 , 22 -RSB- . However , very few randomized studies have evaluated whether psychometric properties and scores differ between the paper and the internet mode of administration of the same health questionnaire -LSB- 23 -- 26 -RSB- . In this study , we compared indicators of the feasibility , reliability , and validity of the CHQ-CF in a subgroup of adolescents who completed the standard paper version of the CHQ-CF with the same indicators in another subgroup of adolescents who completed a newly developed internet version of the questionnaire . Additionally , we compared the mean CHQ-CF scores and distributions of the scale scores between both subgroups . A randomized parallel group design was applied in a large adolescent population -LRB- 13 -- 7 years old -RRB- , ensuring that both subgroups were comparable . The study assessed and compared the paper and internet mode of CHQ-CF administration with regard to the following indicators : the number of missing answers -LRB- indicator of feasibility -RRB- , the distribution of the scale scores including mean scale scores in the whole sample and in gender and age specific subgroups , the internal consistency reliability of multi-item scales -LRB- indicator of reliability -RRB- , the ability of the CHQ-CF to discriminate between subgroups with and without self-reported chronic conditions -LRB- indicator of construct validity -RRB- . Methods Study population In 2003 , 1,071 students in 55 classes of various educational levels in the 3rd year of seven secondary schools -LRB- 13 -- 7 years old -RRB- in the area of Vlaardingen -LRB- metropolitan area -RRB- and Harderwijk -LRB- rural area -RRB- , The Netherlands , were invited to complete the Child Health Questionnaire Child Form -LRB- CHQ-CF -RRB- . The parents and students each received written information about the study several weeks before data collection ; parents could refuse their child 's participation , and participation by the students was voluntary . Data collection The CHQ-CF consists of 87 items with 4 , 5 , or 6 response options divided over 10 multi-item scales and two single-item scales -LRB- Table 1 -RRB- -LSB- 3 -RSB- . To reduce respondent burden , the item `` change-in-health -- was not fielded in this study , and the CHQ-CF scales `` role functioning-emotional -- and `` role functioning-behavioral -- were combined into a single scale . The combination of the two role functioning scales is a departure from the CHQ-CF instructions that makes the test analogous to the parent form of the CHQ in this regard -LSB- 3 -RSB- . For each scale , items were summed up -LRB- some recoded/recalibrated -RRB- and transformed into a 0 -LRB- worst possible score -RRB- to 100 -LRB- best possible score -RRB- scale -LSB- 3 -RSB- . Items on standard socio-demographic variables and the prevalence of seven chronic conditions were included in the questionnaire . From the conventional paper format , using the same wording of the items and instructions , an internet version of the questionnaire was developed through a generic internet tool using PHP -LRB- 4.0.1 -RRB- , MySQL -LRB- 3.22 -RRB- , and JavaScript -LRB- 1.3 -RRB- -LSB- 27 -RSB- . The internet version of the questionnaire listed the items of each CHQ-CF scale on a separate web-page . The internet version did not allow the respondent to select more than one answer to each item of the CHQ-CF and it checked the questionnaire for missing answers before the respondent could `` logout -- If one or more of the items were not answered , the internet version prompted the respondent to go back to complete those items ; but , if the user failed to `` logout -- properly , missing answers would remain.Table 1CHQ-CF scales , items per scale , and interpretation of low and high scoresaCHQ-CF ScalesNumber of itemsDescription low scoreDescription high scorePhysical functioning -LRB- PF -RRB- 9Child is greatly limited in performing all physical activities , including self-care , due to healthChild performs all types of physical activities , including the most vigorous without limitations due to healthRole functioning : Emotional -LRB- RE -RRB- b3Child is greatly limited in school work or activities with friends as a result of emotional problemsChild has no limitations in schoolwork or activities with friends as a result of emotional problemRole functioning : Behavioral -LRB- RB -RRB- b3Child is greatly limited in school work or activities with friends as a result of behavior problemsChild has no limitations in schoolwork or activities with friends as a result of behavior problemsRole functioning : Physical -LRB- RP -RRB- 3Child is greatly limited in school work or activities with friends as a result of physical healthChild has no limitations in schoolwork or activities with friends as a result of physical healthBodily pain -LRB- BP -RRB- 2Child has extremely severe , frequent and limiting bodily painChild has no pain or limitations due to painGeneral behavior -LRB- BE -RRB- 17Child very often exhibits aggressive , immature , delinquent behaviorChild never exhibits aggressive , immature , delinquent behaviorMental health -LRB- MH -RRB- 16Child has feelings of anxiety and depression all of the timeChild feels peaceful , happy , and calm all of the timeSelf esteem -LRB- SE -RRB- 14Child is very dissatisfied with abilities , looks , family/peer relationships and life overallChild is very satisfied with looks , family/peer relationships and life abilities , overallGeneral health perceptions -LRB- GH -RRB- 12Child believes his/her health is poor and likely to get worseChild believes his/her health is excellent and will continue to be soChange in health -LRB- CH -RRB- c1Child 's health is much worse now than 1 year agoChild 's health is much better now than 1 year agoFamily activities -LRB- FA -RRB- 6The child 's health very often limits and interrupts family activities or is a source of family tension.The child 's health never limits or interrupts family activities nor is family a source of tensionFamily cohesion -LRB- FC -RRB- 1Family 's ability to get along is rated `` poor -- Family 's ability to get along is rated `` excellent -- aReproduced with permission -LSB- 3 -RSB- bThe CHQ-CF scales `` Role functioning-emotional -- and `` Role functioning-behavioral -- were merged into a single scale `` Role functioning-emotional/behavioral -- -LRB- REB -RRB- in this studyc This single-item scale was not fielded in this study Randomization Within each school class , students were randomly assigned to either the paper or the internet mode of administration using SPSS-generated random numbers . Students completed the questionnaires , either on paper or online in a classroom with computers linked to the internet , under the supervision of a research assistant ; the students were allowed adequate privacy . Analysis Preparatory secondary vocational education was labelled as `` lower secondary education '' ; secondary schools that prepare students for higher professional training as `` intermediate secondary education '' , and university preparatory secondary education as `` higher secondary education '' . Differences between the characteristics of the participants allocated to the paper versus the internet versions of the questionnaires were tested by Student 's t-test and the χ2 test . We assessed the frequency of missing answers to CHQ-CF items ; the difference in the number of missing answers between the two formats was assessed by the Mann-Whitney U test . We assessed the distributions of the CHQ-CF scale scores to evaluate floor and ceiling effects -LRB- ≥ 25 % of the respondents having the lowest/highest score -RRB- for both modes of administration . Differences between CHQ-CF scale scores by format in the total sample were assessed by Mann-Whitney U tests . Additionally , after transforming the scale scores into ranks , ANOVA was applied to test whether the mode of questionnaire administration interacted with the variables gender -LRB- male n = 432 ; female n = 501 -RRB- and age -LRB- 13 -- 4 year olds , n = 399 ; 15 -- 7 year olds , n = 534 -RRB- . Cohen 's effect sizes , defined as d = -LSB- Mean -LRB- a -RRB- Mean -LRB- b -RRB- -RSB- / SD , where the denominator was the square root of -LSB- -LRB- na-1 -RRB- SDa2 + -LRB- nb-1 -RRB- SDb2 -RSB- / -LSB- -LRB- na-1 -RRB- + -LRB- nb-1 -RRB- -RSB- , were applied to indicate the relative magnitude of score differences between modes of administration . Here , the letters `` a '' and `` b '' refer to the subgroups administered the paper and internet forms of the test , respectively -LSB- 28 -RSB- . Following Cohen 's suggested guidelines , 0.20 ≤ d < 0.50 indicated a `` small effect -- 0.50 ≤ d < 0.80 a `` medium effect -- and d ≥ 0.80 a `` large effect -- -LSB- 28 -RSB- ; Norman et al. have suggested that , in general , d = 0.50 can be considered as threshold for a `` minimally important difference -- -LRB- MID -RRB- -LSB- 29 -RSB- . Cronbach 's α was applied to evaluate the internal consistency reliability of CHQ-CF multi-item scales by format ; α of 0.70 or higher was considered to indicate sufficient internal consistency reliability -LSB- 30 -RSB- . We applied statistical tests of the hypothesis that the Cronbach 's α reliability coefficients of CHQ multi-item scales in the sample administered the test on paper were equal to those administered the test online -LSB- 31 -RSB- . We applied item-level discriminant tests to evaluate whether the CHQ-CF items represent separate domains . For each mode of questionnaire administration , we evaluated whether -LRB- on average -RRB- correlation coefficients -LRB- Pearson-r correlation coefficients -RRB- between the items and their own scale score -LRB- without the item under consideration -RRB- were higher than the correlation coefficients between these items and any other scale . The average Pearson-r correlation coefficients were calculated by applying Fisher 's z transformations -LSB- 32 -RSB- ; we tested whether the differences between the average Pearson-r correlation coefficients in the subgroup administered the paper form and in the subgroup administered the test online were statistically significant -LSB- 33 -RSB- . We assessed the CHQ-CF 's ability to discriminate between subgroups with 0 , 1 or 2 , and 3 or more chronic conditions , after having transformed the CHQ-CF scale scores into ranks , by ANOVA with the independent variables `` number of chronic conditions -- `` mode of questionnaire administration -- and the interaction term `` number of chronic conditions -- `` mode of questionnaire administration -- Cohen 's effect sizes d = -LSB- Mean -LRB- a -RRB- Mean -LRB- b -RRB- -RSB- / SD in the condition subgroup were calculated for 1 or 2 versus 0 conditions , and for ≥ 3 versus 0 conditions . The designations `` a '' and `` b '' refer to the subgroups without chronic conditions and those with chronic conditions , respectively -LSB- 28 -RSB- . All analyses were done using SPSS , Version 11.0.1 . The medical ethical committee of the Erasmus MC-University Medical Center Rotterdam , approved the study . Results Participants and randomization The participation rate was 87 % . The age range of the participants was 13 -- 7 years -LRB- mean age 14.7 years ; SD 0.68 -RRB- , 54 % were female , 93 % were born in the Netherlands , and the majority attended lower secondary education -LRB- Table 2 -RRB- . The prevalence of self-reported chronic conditions was as follows : asthma , 8 % ; allergies , 25 % ; hearing problems , 7 % ; visual problems , 8 % ; headaches or migraine , 17 % ; chronic lower back pain , 17 % ; and depression or anxiety attacks , 8 % -LRB- Table 2 -RRB- . These characteristics were equally distributed in the groups assigned to the paper and internet versions of the questionnaires -LRB- P ≥ 0.05 ; Table 2 -RRB- . The demographic characteristics of the participants -LRB- age , gender , country of birth , and educational level -RRB- reflected those of the general population of Dutch adolescents -LSB- 34 -RSB- . Table 2Characteristics of study participants -LRB- total sample : n = 933 ; participants assigned to the paper questionnaire : n = 475 ; participants assigned to the internet questionnaire : n = 458 -RRB- Total study group -LRB- n = 933 -RRB- Group with paper mode of administration -LRB- n = 458 -RRB- Group with Internet administration -LRB- n = 475 -RRB- P-valueMean -LRB- SD -RRB- or Rangen % of ParticipantsMean -LRB- SD -RRB- or Rangen % of ParticipantsMean -LRB- SD -RRB- or Rangen % of ParticipantsInternet versus paper modeDemographic characteristicsAge -LRB- years -RRB- Mean -LRB- SD -RRB- 14.7 -LRB- 0.68 -RRB- 14.7 -LRB- 0.68 -RRB- 14.7 -LRB- 0.68 -RRB- 0.61 aRange13 -- 713 -- 713 -- 7GenderWomen50154 % 24451 % 25756 % 0.17 bBorn in the NetherlandsYes86693 % 44193 % 42593 % 0.90 bEducational level of the schoolLower secondary education54558 % 27458 % 27159 % Intermediate secondary education17919 % 9420 % 8519 % 0.88 cHigher secondary education20922 % 10723 % 10222 % Chronic conditions : AsthmaYes768 % 408 % 368 % 0.81 bAllergiesYes22925 % 11825 % 11124 % 0.88 bProblems with hearingYes627 % 306 % 327 % 0.70 bProblems with seeingYes788 % 398 % 399 % 0.91 bHeadaches or migraineYes15917 % 8117 % 7817 % 1.00 bChronic low back painYes15917 % 8819 % 7116 % 0.22 bDepression and/or anxiety attacksYes748 % 368 % 388 % 0.72 ba Student 's t-testb Chi square test df = 1c Chi square test df = 2 Difference in the number of missing answers between different modes of CHQ-CF administration At the item level , use of the paper version of the CHQ-CF resulted in more missing answers -LRB- 0 -- .89 % per item ; mean 0.54 % -RRB- compared with the internet version -LRB- 0 -- .22 % per item ; mean 0.04 % ; P < 0.01 -RRB- . CHQ-CF scores by mode of administration A ceiling effect was observed for four CHQ-CF scales in the subgroup that completed the paper questionnaire , and 3 CHQ-CF scales in the subgroup that completed the internet questionnaire -LRB- Table 3 -RRB- . Four CHQ-CF scales , i.e. , `` general behavior -- `` role functioning-physical -- `` mental health -- and `` family activities -- resulted in statistically significant , higher scores for paper versus internet administration -LRB- P < 0.05 -RRB- , but the effect sizes -LRB- d -RRB- were ≤ 0.21 -LRB- Table 3 -RRB- . The mode of questionnaire administration did not interact significantly with gender -LRB- P ≥ 0.05 regarding all scales -RRB- , nor with age -LRB- P ≥ 0.05 regarding six scales -RRB- , except for the CHQ-CF scales `` role functioning-emotional/behavioral -- -LRB- P < 0.05 -RRB- , `` mental health -- -LRB- P < 0.05 -RRB- , `` self esteem -- -LRB- P < 0.05 -RRB- , and `` general health -- -LRB- P < 0.01 -RRB- . Regarding these 4 CHQ-CF scales , administration of the paper version resulted in lower scores than online administration -LRB- or nearly equal scores in the case of `` mental health -- in the subgroup of 13 -- 4 year olds , while in the subgroup of 15 -- 7 year olds , paper administration resulted in higher scores compared with internet administration ; the Cohen 's effect sizes -LRB- d -RRB- for these differences , regardless of sign , were ≤ 0.21 -LRB- data not shown -RRB- . Table 3Comparison of mean scores , distributions of the scale scores , and other psychometric properties of CHQ-CF scales in subgroups with paper -LRB- n = 475 -RRB- and internet modes -LRB- n = 458 -RRB- of questionnaire administrationCHQ-CF scalesa -LRB- range 0 -- 00 -RRB- Mode of admini strationMean -LRB- SD -RRB- Paper versus internet mode of administrationRange of scores % maxd % mine25th % tile50th % tile75th % tileCronbach 's alphaAverage item-own scale correlation fAverage item-other scale correlationP-value -LRB- MWU -RRB- bEffect size dcPhysical FunctioningPaper96 .0 -LRB- 6.9 -RRB- 0.370.0144 -- 00560961001000.690.400.19 Internet95 .8 -LRB- 7.2 -RRB- 37 -- 00530931001000.720.440.18 Role funct.-Emo / behPaper89 .4 -LRB- 17.2 -RRB- 0.140.000 -- 00600781001000.810.650.35 Internet89 .5 -LRB- 15.1 -RRB- 0 -- 00540861001000.70 g0 .51 g0 .28 Role funct.-PhysicalPaper95.0 -LRB- 12.9 -RRB- 0.020.0522 -- 008101001001000.860.740.30 Internet94 .4 -LRB- 11.7 -RRB- 22 -- 00750891001000.76 g0 .60 g0 .25 Bodily PainPaper73 .5 -LRB- 22.7 -RRB- 0.56 -- 0.050 -- 0025060801000.880.800.35 Internet74 .7 -LRB- 21.4 -RRB- 0 -- 002416080900.890.810.30 General BehaviorPaper80 .9 -LRB- 10.6 -RRB- 0.000.2125 -- 00107582880.830.440.26 Internet78 .6 -LRB- 11.4 -RRB- 35 -- 00007279870.850.460.26 Mental HealthPaper76 .5 -LRB- 15.4 -RRB- 0.010.1214 -- 00206980890.920.630.35 Internet74 .6 -LRB- 14.8 -RRB- 14 -- 00006777840.90 h0 .600.31 Self EsteemPaper74 .7 -LRB- 12.2 -RRB- 0.990.0220 -- 00106875820.880.560.30 Internet74 .4 -LRB- 11.6 -RRB- 25 -- 00007076820.870.550.28 General HealthPaper73 .5 -LRB- 16.5 -RRB- 0.590.0519 -- 00306376860.820.500.28 Internet72 .6 -LRB- 16.7 -RRB- 20 -- 00106375860.810.580.28 Family ActivitiesPaper80 .0 -LRB- 17.7 -RRB- 0.030.118 -- 001706783960.810.580.24 Internet78 .1 -LRB- 16.9 -RRB- 25 -- 001306779920.780.540.24 Family CohesionPaper70 .6 -LRB- 23.5 -RRB- 0.910.000 -- 00182608585nana0 .36 Internet70 .7 -LRB- 23.0 -RRB- 0 -- 00162608585nana0 .33 aThe CHQ-CF scales ` Role functioning-emotional -- and ` Role functioning-behavioral -- were merged into a single scale in this studybTwo-sided Mann-Whitney U test of scale scores of the group using paper , and the group using the internet version of the questionnairecDifference of mean scores divided by weighted average of SDs in groups given the paper and internet version of the questionnaires -LSB- 28 , 29 -RSB- d/e % of respondents with best/worst possible score -LRB- ceiling/floor -RRB- fEach item was correlated with the applicable ad-hoc scale without the item under considerationg/hStatistically significant differences of Cronbach 's alpha/average Pearson-r correlation coefficients in subgroups given paper or internet questionnaires -LRB- gP ≤ 0.01 ; hP ≤ 0.05 -RRB- -LSB- 31 , 33 -RSB- . na Not applicable ; Role funct.-Emo / beh - Role functioning-emotional behavioral ; Role funct.-Physical - Role functioning-physical Internal consistency reliability of scales by mode of administration Cronbach 's αs for the two formats were adequate for all CHQ-CF scales , except `` physical functioning -- in the subgroup administered the paper version of the questionnaire -LRB- α = 0.69 -RRB- . The two `` role functioning -- scales and `` mental health -- showed statistically significant , higher Cronbach 's αs in the subgroup administered the paper version of the questionnaire compared with the alphas in the subgroup administered the internet version -LRB- P < 0.01 , respectively P < 0.05 -RRB- -LRB- Table 3 -RRB- . All multi-item scales , regarding both modes of administration , showed higher average -LRB- corrected -RRB- item-own scale correlation coefficients than average item-other-scale correlation coefficients . The two `` role functioning -- scales showed statistically significant , higher average item-own scale correlation coefficients in the subgroup administered the paper version of the questionnaire compared with the item-own scale correlation coefficients in the subgroup administered the internet version -LRB- P < 0.01 -RRB- -LRB- Table 3 -RRB- . Construct validity by mode of administration All mean CHQ-CF scale scores were lower in the subgroup with one or two reported conditions and in the subgroup with three or more reported conditions when either was compared with the subgroup with no reported conditions . For both modes of questionnaire administration , and for all CHQ-CF scales , the more chronic conditions that were reported , the higher the effect sizes compared with the subgroup with no chronic conditions . ANOVA showed statistically significant CHQ-CF score differences by `` number of chronic conditions -- for all scales -LRB- P < 0.01 -RRB- -LRB- Table 4 -RRB- . The mode of questionnaire administration did not interact significantly with the variable `` number of chronic conditions -- -LRB- P ≥ 0.05 for all scales -RRB- -LRB- Table 4 -RRB- . Table 4Ability of the CHQ-CF scales to discriminate between subgroups differing in the participants -- number of chronic conditions , for the group that was assigned to complete the paper version -LRB- n = 475 -RRB- and for the group that was assigned to complete the internet version -LRB- n = 458 -RRB- Number of chronic conditions per participantCHQ-CF scalesa -LRB- range 0 -- 00 -RRB- : Mode of admini - stration0 conditions1 or 2 conditions ≥ conditions1 or 2 versus 0 conditions ≥ versus 0 conditionsANOVA P-value Number of chronic conditionsANOVA P-value Internet versus paper modeANOVA P-value Interaction term Number of chronic conditions by mode of questionnaire administrationn = 224 -LRB- Paper -RRB- n = 206 -LRB- Internet -RRB- Mean -LRB- SD -RRB- n = 210 -LRB- Paper -RRB- n = 217 -LRB- Internet -RRB- Mean -LRB- SD -RRB- n = 41 -LRB- Paper -RRB- n = 35 -LRB- Internet -RRB- Mean -LRB- SD -RRB- Effect size dbEffect size dbPhysical functioningPaper97 -LRB- 5 -RRB- 96 -LRB- 7 -RRB- 90 -LRB- 10 -RRB- 0.240.80 * Internet97 -LRB- 5 -RRB- 95 -LRB- 7 -RRB- 89 -LRB- 12 -RRB- 0.280.64 * 0.000.950.52 Role funct.-emo / behavPaper93 -LRB- 13 -RRB- 88 -LRB- 18 -RRB- 77 -LRB- 24 -RRB- 0.250.64 * Internet91 -LRB- 14 -RRB- 89 -LRB- 15 -RRB- 79 -LRB- 18 -RRB- 0.150.66 * 0.000.200.72 Role funct.-physicalPaper97 -LRB- 11 -RRB- 94 -LRB- 13 -RRB- 91 -LRB- 17 -RRB- 0.190.34 Internet96 -LRB- 10 -RRB- 94 -LRB- 13 -RRB- 91 -LRB- 13 -RRB- 0.140.340.000.070.69 Bodily painPaper82 -LRB- 18 -RRB- 70 -LRB- 22 -RRB- 47 -LRB- 24 -RRB- 0.52 * 1.45 * Internet80 -LRB- 18 -RRB- 72 -LRB- 21 -RRB- 55 -LRB- 24 -RRB- 0.391.03 * 0.000.200.32 General behaviorPaper84 -LRB- 9 -RRB- 80 -LRB- 10 -RRB- 71 -LRB- 13 -RRB- 0.400.98 * Internet81 -LRB- 10 -RRB- 78 -LRB- 11 -RRB- 69 -LRB- 14 -RRB- 0.260.90 * 0.000.030.65 Mental healthPaper83 -LRB- 10 -RRB- 74 -LRB- 16 -RRB- 57 -LRB- 16 -RRB- 0.56 * 1.58 * Internet80 -LRB- 11 -RRB- 72 -LRB- 15 -RRB- 59 -LRB- 18 -RRB- 0.501.20 * 0.000.260.29 Self esteemPaper79 -LRB- 10 -RRB- 73 -LRB- 12 -RRB- 61 -LRB- 12 -RRB- 0.52 * 1.44 * Internet77 -LRB- 9 -RRB- 73 -LRB- 12 -RRB- 66 -LRB- 14 -RRB- 0.330.80 * 0.000.260.06 General healthPaper80 -LRB- 14 -RRB- 71 -LRB- 15 -RRB- 54 -LRB- 14 -RRB- 0.57 * 1.82 * Internet79 -LRB- 13 -RRB- 70 -LRB- 17 -RRB- 55 -LRB- 18 -RRB- 0.54 * 1.29 * 0.000.740.58 Family activitiesPaper83 -LRB- 16 -RRB- 79 -LRB- 18 -RRB- 70 -LRB- 20 -RRB- 0.260.67 * Internet81 -LRB- 16 -RRB- 78 -LRB- 17 -RRB- 66 -LRB- 15 -RRB- 0.170.97 * 0.000.030.51 Family cohesionPaper77 -LRB- 21 -RRB- 68 -LRB- 23 -RRB- 53 -LRB- 26 -RRB- 0.380.89 * Internet74 -LRB- 20 -RRB- 68 -LRB- 24 -RRB- 64 -LRB- 28 -RRB- 0.240.380.000.180.05 a The CHQ-CF scales ` Role functioning-emotional -- and ` Role functioning-behavioral -- were merged into a single scale in this studybDifference of the means divided by SD in the subgroup with chronic condition -LRB- s -RRB- , where * indicates at least a `` minimally important difference -- -LRB- d ≥ 0.50 -RRB- -LSB- 28 , 29 -RSB- Role funct.-Emo / beh - Role functioning-emotional behavioral ; Role funct.-Physical - Role functioning-physical Discussion and conclusions In this study we applied a randomized design to compare the results of the Child Health Questionnaire-Child Form -LRB- CHQ-CF -RRB- administered by a paper questionnaire and by an online questionnaire . The results provided support for the feasibility , internal consistency reliability , and construct validity of the CHQ-CF scales . Both modes of questionnaire administration yielded comparable scale scores and showed comparable psychometric properties . Additionally , the study provided reference/norm scores for clinical studies -LRB- general population of 13 -- 7 year olds -RRB- . Strengths of the current study The participation rate was high . Study group characteristics -LRB- age , gender , country of birth , and educational levels -RRB- were representative of those of the general population of Dutch adolescents -LSB- 34 -RSB- . Randomization to either the paper or internet mode of administration of the CHQ-CF was successful with respect to the evaluated characteristics . Limitations We applied a randomized parallel group design that allows for the comparison of indicators of feasibility , reliability , and validity at the group level between a subgroup that completed a paper version and a subgroup that completed an internet version of the CHQ-CF . However , this did not allow an evaluation of whether the same person would provide equivalent or different answers to the same CHQ-CF questionnaire administered by the alternative mode , which would require a randomized crossover design -LSB- 25 , 35 -RSB- . Such an evaluation at the individual level requires the respondent to forget all previously provided answers at the second assessment , e.g. , by waiting 1 or 2 weeks between the two measurements . It also requires that there is no effect from having previously completed a CHQ-CF questionnaire by any mode , paper or internet , at the second assessment , and that scores by the same mode of administration after a relatively short interval , in the absence of changes in health status , are exactly the same . However , in an evaluation of retesting with the same paper version of the CHQ-CF after 2 weeks , 5 out of 10 CHQ-CF scales showed statistically significant , higher scores at the second measurement with Cohen 's effect sizes ranging from 0.25 to 0.40 , while intraclass correlation coefficients between the first and second measurement ranged from 0.06 thru 0.84 -LSB- 7 -RSB- . Furthermore , in a randomized crossover design , `` carry-over -- effects may be present , i.e. , completing an internet version before a paper version may have a different effect on the second assessment , than does completing a paper version before an internet version -LSB- 35 -RSB- . Despite the logistical and the above-mentioned methodological challenges , we recommend future studies comparing the paper and internet versions of the CHQ-CF applying a randomized crossover design to evaluate congruency of answers to CHQ-CF items at the individual level . In this study , internet and paper questionnaires were completed in a controlled environment with adequate privacy and supervision . This may not be the case during future applications . We are unaware of the impact less privacy during completion of the questionnaires may have , but this would apply to both the paper and the internet versions of the questionnaire . For both modes of questionnaire administration , we did not evaluate correlations between CHQ-CF scores and a relevant parent-rated questionnaire such as the CHQ-PF50 -LSB- 2 , 3 -RSB- . Test-retest reliability of the CHQ-CF and its responsiveness and sensitivity to changes in health were not evaluated in the current study . The CHQ-CF has a relatively large number of items ; therefore , we recommend developing a shorter version in the future . Psychometric properties The psychometric properties , with only a few exceptions , were equal between the two modes of questionnaire administration . The Cronbach 's α of the scale `` physical functioning -- in the subgroup administered the paper version of the questionnaire was just under 0.70 , and the difference with the alpha in the subgroup administered the internet version was not statistically significant . Missing values Compared with the paper version , the internet version was successful in reducing the quantity of missing data . Theoretically , differences in selective partial non-response between formats might have contributed to differences in scores that were reported in this study . In our study , in the subgroup -LRB- n = 86 -RRB- that had at least one missing answer to a paper CHQ-CF item , all scale means were somewhat lower than in the subgroup -LRB- n = 389 -RRB- with no missing answers , but these differences were not significant -LRB- P ≥ 0.05 -RRB- . Thus , missing answers are not a source of the observed score differences . Score differences between modes of questionnaire administration Recently , Ritter et al. found no statistically significant score differences between internet and paper modes of administration for 16 health-related measures , but the study was conducted in an opportunity sample retrieved from the internet , which limits its generalizability -LSB- 23 -RSB- . In a randomized internet-paper comparison among adolescents concerning various health measures other than the CHQ-CF , only one statistically significant score difference was reported among 21 topics -LSB- 24 -RSB- . In another randomized adolescent study , a medical consumption index and 11 indicators of fruit consumption and determinants of fruit consumption did not show statistically significant score differences between internet and paper administration of the questionnaire , except for one measure that showed small score differences between modes of administration -LSB- 25 -RSB- . The International Study of Asthma and Allergies in Childhood -LRB- ISAAC -RRB- questionnaire did not show statistically significant score differences between internet and paper administration in two randomized adolescent studies -LSB- 25 , 26 -RSB- . In our study , in the whole sample , the paper version resulted in slightly , yet statistically significant , higher scores for 4 of 10 CHQ-CF scales compared with the internet version . One plausible explanation is chance , since it should be considered that given multiple comparisons , there is a 1-in-20 chance of a false association for each comparison -LRB- Type I error at α = 0.05 -RRB- -LSB- 36 -RSB- . A commonly used Bonferroni correction for 10 comparisons would imply an adjusted α = 0.05 / 10 = 0.005 -LSB- 36 -RSB- ; at α = 0.005 , only one score difference -LRB- regarding the scale `` general behavior -- was significant . Furthermore , given Cohen 's suggested guidelines for the interpretation of effect sizes , three of the four statistically significant differences between modes of administration can be considered as negligible -LRB- d ≤ 0.12 -RRB- , and one difference regarding the CHQ-scale `` general behavior -- -LRB- d = 0.21 -RRB- can be considered as small -LSB- 28 -RSB- ; all effect sizes were far below d = 0.50 that was suggested as an approximate threshold for `` minimally important differences -- by Norman et al. -LSB- 29 -RSB- . This study provides no explanations for the established small score differences between paper and internet administration , or for the established statistically significant , but small interaction effects of administration mode with age in the case of four CHQ-CF scales . Conclusions With increasing application of online health questionnaires rather than questionnaires on paper , especially in adolescent populations , it should be noted that comparison of results requires that the scores between these modes of administration do not show meaningful statistically significant differences . This study showed that , overall , paper and internet versions of the CHQ-CF yielded only a few , negligible or small , differences . Paper and internet modes of CHQ-CF administration may be combined in a single study , although researchers should consider the possibility of minor score differences depending on the mode of administration for some scales . We recommend repeated studies in other populations , including clinical populations , to confirm or reject our results .

B O B O O B O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O B O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

health status measurement
adolescents
feasibility
reliability
validity
online questionnaire
internet questionnaire

child health questionnaire child form 87 items (chq-cf87)


P2 receptors in atherosclerosis and postangioplasty restenosis Atherosclerosis is an immunoinflammatory process that involves complex interactions between the vessel wall and blood components and is thought to be initiated by endothelial dysfunction -LSB- Ross -LRB- Nature 362:801 -- 09 , 1993 -RRB- ; Fuster et al. -LRB- N Engl J Med 326:242 -- 50 , 1992 -RRB- ; Davies and Woolf -LRB- Br Heart J 69 : S3 -- S11 , 1993 -RRB- -RSB- . Extracellular nucleotides that are released from a variety of arterial and blood cells -LSB- Di Virgilio and Solini -LRB- Br J Pharmacol 135:831 -- 42 , 2002 -RRB- -RSB- can bind to P2 receptors and modulate proliferation and migration of smooth muscle cells -LRB- SMC -RRB- , which are known to be involved in intimal hyperplasia that accompanies atherosclerosis and postangioplasty restenosis -LSB- Lafont et al. -LRB- Circ Res 76:996 -- 002 , 1995 -RRB- -RSB- . In addition , P2 receptors mediate many other functions including platelet aggregation , leukocyte adherence , and arterial vasomotricity . A direct pathological role of P2 receptors is reinforced by recent evidence showing that upregulation and activation of P2Y2 receptors in rabbit arteries mediates intimal hyperplasia -LSB- Seye et al. -LRB- Circulation 106:2720 -- 726 , 2002 -RRB- -RSB- . In addition , upregulation of functional P2Y receptors also has been demonstrated in the basilar artery of the rat double-hemorrhage model -LSB- Carpenter et al. -LRB- Stroke 32:516 -- 22 , 2001 -RRB- -RSB- and in coronary artery of diabetic dyslipidemic pigs -LSB- Hill et al. -LRB- J Vasc Res 38:432 -- 43 , 2001 -RRB- -RSB- . It has been proposed that upregulation of P2Y receptors may be a potential diagnostic indicator for the early stages of atherosclerosis -LSB- Elmaleh et al. -LRB- Proc Natl Acad Sci U S A 95:691 -- 95 , 1998 -RRB- -RSB- . Therefore , particular effort must be made to understand the consequences of nucleotide release from cells in the cardiovascular system and the subsequent effects of P2 nucleotide receptor activation in blood vessels , which may reveal novel therapeutic strategies for atherosclerosis and restenosis after angioplasty . Introduction Atherosclerosis is a pathological phenomenon primarily affecting the large conduit arteries , for example the aorta , coronary , carotid iliac , and femoral arteries . The development of atherosclerotic lesions in arteries involves the intimal recruitment of smooth muscle cells -LRB- SMC -RRB- within the blood vessel wall , and also the infiltration of blood-derived cells -LSB- 1 -RSB- . This process necessitates the proliferation and migration of SMC from the underlying media and the endothelial adhesion of leukocytes and their infiltration into the subendothelium . A similar intimal accumulation of SMC also takes place during the postangioplasty restenotic process . Although the factors involved in intimal cell recruitment are not clearly identified , it is becoming evident that endothelial dysfunction is a key factor in the development of vascular disease . Experimental evidence suggests that an intact endothelium plays a central role in maintaining a low proliferative state of SMC under normal conditions -LSB- 10 -RSB- . In arterial injury , endothelial cells , SMC , and various blood cells can release chemotactic factors and mitogens , including ATP and other nucleotides -LRB- see -LSB- 4 -RSB- for review -RRB- . Activation of P2 nucleotide receptors has been shown to induce not only the proliferation and migration of vascular SMC but also apoptosis , a process involved in the evolution of the atherosclerotic plaque -LSB- 11 -RSB- . In addition , P2 receptors mediate both vasorelaxation and vasoconstriction of arteries that may be involved in the vascular remodeling accompanying atherosclerosis and postangioplasty restenosis -LSB- 5 -RSB- . A better understanding of the causative agents and mechanisms of proliferation and migration of vascular SMC as well as recruitment of blood-derived cells by the endothelium could lead to prevention , attenuation , or even reversal of intimal thickening , which may dramatically reduce morbidity and mortality from vascular diseases such as atherosclerosis and restenosis after angioplasty . In this respect , a better understanding of the physiological role of P2 receptors in both normal and pathological blood vessels could potentially lead to a breakthrough in the fight against vascular disease . P2 receptors in the cardiovascular system Extracellular nucleotides bind to cell surface receptors known as P2 receptors , which are present in many tissues . To date , these receptors have been classified into two main families : the P2X receptors that are ligand-gated ion channels comprised of homo - or hetero-oligomers -LSB- 12 -RSB- and P2Y receptors that are seven membrane spanning receptors coupled via G proteins -LRB- Gq/11 or Gi/o -RRB- to phospholipase C -LRB- PLC -RRB- and/or adenylate cyclase -LSB- 12 -- 14 -RSB- . In turn , PLC activation generates inositol 1,4,5-triphosphate -LRB- IP3 -RRB- , a mediator of Ca2 + release from intracellular stores , and diacylglycerol , an activator of protein kinase C -LRB- PKC -RRB- , whereas adenylate cyclase generates cyclic adenosine monophosphate -LRB- cAMP -RRB- , an activator of protein kinase A -LRB- PKA -RRB- . The cloning of seven P2X -LRB- P2X1 , P2X2 , P2X3 , P2X4 , P2X5 , P2X6 , P2X7 -RRB- and eight P2Y -LRB- P2Y1 , P2Y2 , P2Y4 , P2Y6 , P2Y11 , P2Y12 , P2Y13 , P2Y14 -RRB- receptor subtypes has made it possible to use molecular and pharmacological approaches to study the distribution and functional properties of specific P2 receptor subtypes at the tissue and cellular level . P2 receptors in vascular cells The normal arterial wall consists of three layers : the intima , media , and adventitia . The single layer of endothelial cells facing the vessel lumen is a very important component of the vascular wall in terms of releasing both vasodilators such as nitric oxide -LRB- NO -RRB- and prostacyclin -LRB- PGI2 -RRB- and vasoconstrictors like thromboxane A2 and endothelin . The principal P2Y receptor subtypes that have been functionally characterized in endothelial cells are P2Y1 and P2Y2 , but mRNAs for P2Y4 and P2Y6 receptors have also been detected -LSB- 4 -RSB- . Endothelium-dependent vasorelaxation has been attributed to the release of NO and PGI2 after binding of nucleotides to P2Y1 and P2Y2 receptors in endothelial cells -LSB- 15 -RSB- , whereas vasoconstrictor effects in SMC result from the action of nucleotides on P2Y2 and P2X receptors -LSB- 16 , 17 -RSB- . In most blood vessels , P2Y1 receptors for ADP are present on the endothelium and regulate vasodilatation by Ca2 + - dependent -LRB- PLC-mediated -RRB- activation of nitric oxide synthase -LRB- NOS -RRB- and generation of endothelial-dependent relaxing factor -LRB- EDRF -RRB- -LRB- see -LSB- 4 -RSB- for review -RRB- . Endothelial prostacyclin production is also stimulated by P2Y1 and P2Y2 receptors , but this seems to play a minimal role in vasodilatation , at least under physiological conditions -LRB- see -LSB- 18 -RSB- for review -RRB- . Recent studies have indicated that in the aorta of P2Y2-null mice the endothelium-dependent relaxation by ATP and ATPγS was inhibited , demonstrating the role of the P2Y2 receptors , but that a relaxation by UTP and UDP was maintained , suggesting the additional involvement of P2Y6 receptors -LSB- 19 -RSB- . The majority of the cells in intact blood vessels are SMC , which occupy most of the media , and are involved in vasoconstriction and vasorelaxation of the vessel . P2Y2Rs in SMC mediate the induction of immediate-early and delayed-early cell cycle-dependent genes , consistent with a role for P2Y2Rs in vascular proliferation of SMC -LSB- 20 , 21 -RSB- . A recent study demonstrated that P2Y2 is the predominant functional receptor that responds to ATP and UTP in rat aortic SMC -LSB- 22 -RSB- . In human cerebral arteries , P2Y6 seems to be the predominant subtype and induces vasoconstriction when activated by UDP/UTP -LSB- 23 , 24 -RSB- . This is consistent with findings from rat pulmonary and mesenteric arteries -LSB- 25 , 26 -RSB- . In addition , a recent study on P2X1 knockout mice further supports the prominent contractile effect of the P2Y6 subtype in mesenteric arterial trees -LSB- 27 -RSB- . Taken together , it seems that the principal receptor mediating UTP/UDP-induced contractile responses in blood vessels might be the P2Y6 subtype . Other studies have reported the presence of both P2Y4 and P2Y6 receptors in rat aortic SMC -LSB- 21 , 28 , 29 -RSB- . P2Y1 receptors are expressed in SMC of a number of blood vessel types and like their EC counterparts mediate vasodilatation most likely through the activation of K + channels -LRB- see -LSB- 18 -RSB- for review -RRB- . The presence of several P2X receptor subtypes also has been reported in human saphenous vein SMC , including P2X1 , P2X2 , P2X4 , and P2X7 receptors -LSB- 30 -RSB- . The outermost layer of the blood vessel consists of connective tissue and fibroblasts , which have not been appreciated in the regulation of vascular tone . A recent study showed that fibroblasts can migrate into the neointima , suggesting their possible involvement in the development of vascular diseases such as atherosclerosis and restenosis after angioplasty -LSB- 31 -RSB- . Since human and rat fibroblasts are known to express P2Y1 , P2Y2 , P2Y4 , P2Y6 , P2X3 , P2X4 , and P2X7 receptors -LSB- 32 -RSB- , further investigation is needed to establish the role of fibroblast P2 receptors in either physiological or pathophysiological conditions . P2 receptors in blood cells G protein-coupled P2Y receptors whose activation leads to intracellular calcium mobilization have been observed in neutrophils -LSB- 4 , 33 , 34 -RSB- , and turkey erythrocytes express both P2Y1 and P2X7 receptors -LSB- 35 , 36 -RSB- . ATP and UTP act as secretagogues by binding to P2Y2Rs to enhance exocytosis of primary granules in neutrophils -LSB- 37 -RSB- . In macrophages , ATP activates a P2X7 receptor that differs from other ligand-gated ion channel P2X receptors by its ability to generate plasma membrane pores when activated -LSB- 38 -RSB- . Human T lymphocytes also have been shown to express P2X7 receptors -LSB- 39 -RSB- . Human monocytes and macrophages coexpress P2X1 , P2X4 , and P2X7 receptors , whereas granulocytes only express P2X7 receptors -LSB- 40 -RSB- . P2Y1 , P2Y2 , and P2Y6 receptors also are expressed in monocytes , B lymphocytes , and polymorphonuclear granulocytes -LSB- 41 -RSB- . Human platelets express P2Y1 , P2Y12 , and P2X1 receptors -LSB- 42 -- 44 -RSB- . Thus , the diversity of P2 receptor expression in blood cells , as well as endothelial cells , SMC , and fibroblasts , suggests that this receptor superfamily plays a significant role in the regulation of cardiovascular functions . P2 receptors regulate nucleotide-induced vascular smooth muscle cell proliferation Proliferation of SMC is a hallmark of vascular diseases such as atherosclerosis and restenosis following angioplasty . ATP and other nucleotides are released by aggregating platelets and damaged vascular cells , such as endothelial cells and SMC in pathological or stress conditions -LSB- 45 -RSB- . Extracellular nucleotides can act via P2X and P2Y receptors to induce acute responses such as the regulation of vascular tone -LSB- 46 -RSB- . However , it is generally believed that the ionotropic P2X receptors do not mediate the chronic responses of nucleotides such as cell proliferation . The mitogenic effect of extracellular nucleotides on vascular SMC -LRB- VSMC -RRB- has been known for years -LSB- 47 -RSB- . However , a potent antiproliferative effect of UTP on VSMC also has been reported in human arterial and venous SMC -LSB- 48 -RSB- . In either case , the P2 receptor subtype -LRB- s -RRB- responsible for these effects on the proliferation of VSMC has not been determined . Earlier studies have shown that ATP or UTP increased DNA and protein synthesis in subcultured rat aortic VSMC -LSB- 49 , 50 -RSB- . In the same cell culture model , however , Malam-Souley et al. -LSB- 51 -RSB- were unable to detect increases in DNA synthesis after ATP/UTP stimulation , although ATP or UTP upregulated the expression of mRNA to several cell cycle progression-related genes . Because P2X receptor agonists were essentially inactive , it was concluded that a P2U-like receptor -LRB- now termed P2Y2 -RRB- was responsible for the mitogenic effects of ATP/UTP . However , the role of a P2Y4 receptor can not be excluded , because the nucleotide agonist profile between rat P2Y2 and P2Y4 receptors is essentially indistinguishable -LSB- 52 -RSB- . Indeed , Harper et al. -LSB- 53 -RSB- suggested that the P2Y4 receptor mediated ATP/UTP-induced proliferation of rat aortic VSMC . Recent studies indicated that ATP , UTP , or ITP , three agonists of the cloned porcine P2Y2 receptor , increased DNA and protein synthesis and cell number in coronary artery SMC -LSB- 54 -RSB- . Indeed , treatment of pig coronary artery SMC with UTP , ATP , or ITP caused a concentration-dependent increase in DNA and protein synthesis and cell number , whereas UDP only caused a small increase in protein synthesis . Intriguingly , ATP was much more potent and efficacious than UTP , ITP , and UDP in increasing DNA synthesis and expression of PCNA , a protein marker of cell proliferation , suggesting that another receptor may contribute to the proliferative response -LSB- 54 -RSB- . In vivo experiments have shown that intimal thickening of collared rabbit carotid arteries was greatly enhanced by in situ UTP application and was closely associated with osteopontin expression in medial SMC -LSB- 6 -RSB- . Osteopontin is chemotactic for SMC and is associated with arterial smooth muscle cell proliferation -LSB- 55 -RSB- . Moreover , both UTP and ATP increased osteopontin expression in cultured SMC , whereas ADP , UDP , and 2-MeSATP were ineffective , which suggests a role for the P2Y2R in which ATP and UTP are equipotent . Direct evidence for involvement of the P2Y2R was provided by inhibition of UTP-induced osteopontin expression in cultured SMC by P2Y2 antisense oligonucleotides -LSB- 6 -RSB- . P2Y6 receptors also have been shown to mediate proliferation of SMC in rat aorta -LSB- 56 -RSB- . Role of P2 receptors in the migration of vascular SMC Recent studies indicate that the extracellular nucleotides ATP , ADP , UTP , and UDP serve as directional cues for the migration of rat aortic SMC -LSB- 57 -RSB- . At identical concentrations , the most powerful migratory response induced by these nucleotides was elicited by UTP . Nucleotide-induced migration of SMC is the consequence of both chemotaxis and chemokinesis and may result either from the activation of one particular P2 nucleotide receptor subtype or from activation of several P2 receptor subtypes . The ability of UTP at submicromolar levels to stimulate migration of SMC supports the hypothesis that this response could have physiological consequences and is essentially mediated by P2Y2 receptor activation without excluding participation of other P2Y receptor subtypes . The difference in the capacity of UTP and ATP to elicit migration of SMC could be due to the inhibition of nucleotide-induced cell migration by adenosine generated from ATP catabolism by cell surface ectonucleotidases . Indeed , ATP and UTP were equally effective in causing migration of SMC when ATP was prevented from degradation by addition of the ectonucleotidase inhibitor α,β-methylene-ATP -LSB- 57 -RSB- . Several P2Y receptor subtypes could be involved in nucleotide-induced migration of SMC . It has been shown in rat aortic SMC that the P2Y2 receptor is the predominant P2Y receptor subtype -LSB- 28 , 58 -RSB- , whereas lower levels of P2Y4 and P2Y1 receptor mRNA were detected -LSB- 58 -RSB- . The very low level of P2Y1 receptor mRNA expression was consistent with the absence of ADP-induced migration of cultured rat aortic SMC , demonstrating that P2Y1 is not involved in this process -LSB- 58 -RSB- . In addition , the same study showed that a commercially available solution of hexokinase-treated UDP -LRB- UTP-free -RRB- induced cell migration equally well as untreated UDP , thereby demonstrating that UDP is chemotactic for aortic SMC by activation of the P2Y6 receptor . Conversely , migration of rat aortic SMC induced by UTP occurred even when UTP degradation by nucleoside diphosphate kinase was inhibited , demonstrating the involvement of P2Y2 and/or P2Y4 receptor -LRB- s -RRB- -LSB- 58 -RSB- . The increased migration of SMC in response to extracellular nucleotides could be related to increases in extracellular matrix -LRB- ECM -RRB- protein expression . Indeed , previous studies have shown that UTP induces osteopontin expression in rat and rabbit aortic SMC -LSB- 51 , 6 -RSB- . Increased expression of osteopontin , an RGD-containing ECM protein , is associated with the activation of rat arterial SMC in vitro and in vivo -LSB- 57 -RSB- . The increase in osteopontin expression plays a key role in UTP-induced migration of rat aortic SMC , since a monoclonal antibody against osteopontin fully abolished UTP-induced migration -LSB- 57 -RSB- , whereas an antibody against vitronectin , another ECM protein also involved in migration of human SMC -LSB- 59 -RSB- , had no effect on the migration of rat aortic SMC -LSB- 57 -RSB- . UTP induces increases in osteopontin mRNA expression by increasing both osteopontin mRNA stabilization and osteopontin promoter activity -LSB- 60 -RSB- . Recent studies have shown that activation of an AP-1 binding site located 76 bp upstream of the transcription start in the rat osteopontin promoter is involved in UTP-induced osteopontin expression . Using a luciferase promoter deletion assay , Renault et al. -LSB- 61 -RSB- identified a new region of the rat osteopontin promoter -LRB- -1837 to -1757 -RRB- that is responsive to UTP . This region contains an NFκB site located at -1800 and an Ebox located at -1768 . Supershift electrophoretic mobility shift and chromatin immunoprecipitation assays identified NFκB and USF-1 / USF-2 as DNA binding proteins induced by UTP . Using dominant negative mutants of IκB kinase and USF transcription factors , it was confirmed that NFκB and USF-1 / USF-2 are involved in the UTP-induced expression of osteopontin . This ability of nucleotides to act as chemoattractants for rat arterial SMC in a concentration range potentially found in pathological vessels -LSB- 62 -RSB- and the findings of previous studies demonstrating the mitogenic activity of extracellular nucleotides for these cells suggest that nucleotides released from mechanically stretched vascular or damaged cells during the angioplasty process may participate in arterial wall remodeling . Role of P2 receptors in nucleotide-induced vascular inflammation In addition to their mitogenic effects , extracellular nucleotides may also cause cell recruitment by inducing lymphocyte and macrophage adhesion to human pulmonary artery endothelial cells as demonstrated in vitro -LSB- 63 -RSB- . Nucleotides can also modulate rat aortic smooth muscle cell adhesion and migration by increasing the expression of osteopontin -LSB- 51 , 57 -RSB- , a protein involved in both processes . Moreover , extracellular nucleotides may play a role in intra-arterial attraction of monocytes by inducing an increased expression of monocyte-chemoattractant protein-1 by arterial SMC -LSB- 21 -RSB- . Stimulation of P2 receptors is coupled to the release of the proinflammatory cytokines interleukin -LRB- IL -RRB- -1 β , IL-1α , IL-8 , and tumor necrosis factor -LRB- TNF -RRB- - α -LRB- see -LSB- 4 -RSB- for review -RRB- that are of obvious relevance to inflammation in atherosclerosis . Activation of P2X7 receptors on monocytes/macrophages enhances release of proinflammatory cytokines that modulate NO production and expression of inducible nitric oxide synthase -LRB- iNOS -RRB- -LRB- see -LSB- 64 -RSB- for review -RRB- , mediators of immune cell activation that is an early step in atherosclerotic lesion development . Monocyte recruitment into the vessel wall is a complex process that includes cell rolling , firm attachment , and directed migration . It is now becoming evident that adhesion molecules such as VCAM-1 play an important role in leukocyte adherence to vascular endothelial cells -LSB- 65 , 66 -RSB- . VCAM-1 expression is induced or upregulated by proinflammatory cytokines such as TNF-α and IL-1β in cellular components of the arterial wall including endothelial cells , smooth muscle cells , and fibroblasts -LSB- 67-69 -RSB- . ATP and UTP have been shown to induce cell-cell adhesion in a human monocyte/macrophage lineage and neutrophil adherence to human endothelial cell monolayers -LSB- 63 , 70 -RSB- . Recent studies have shown that local UTP delivery via an osmotic pump to collared rabbit carotid arteries induced intimal accumulation of macrophages , similar to oxidized low-density lipoprotein -LRB- LDL -RRB- , a response that was mediated by activation of P2Y2 receptors -LSB- 6 -RSB- . Leukocyte migration depends on the activities of adhesion proteins -LRB- e.g. , selectins and integrins -RRB- on leukocytes and vascular endothelial cells . We have demonstrated that activation of P2Y2 receptors in endothelial cells causes the expression of VCAM-1 that mediates the adherence of monocytes to vascular endothelium -LSB- 71 -RSB- , leading to their penetration into the vessel wall to promote arterial inflammation associated with atherosclerosis . Recent studies have revealed that a Src homology-3 -LRB- SH3 -RRB- binding domain in the C-terminal tail of the P2Y2 receptor promotes the nucleotide-induced association of Src with the P2Y2 receptor , leading to the transactivation of growth factor receptors , such as the epithelial growth factor -LRB- EGF -RRB- and vascular endothelial growth factor -LRB- VEGF -RRB- receptors , and nucleotide-induced upregulation of VCAM-1 -LSB- 72 , 73 -RSB- . Since leukocyte infiltration and migration are key processes involved in atherosclerosis , these findings suggest that P2Y2 receptors represent a novel target for reducing arterial inflammation associated with cardiovascular disease . P2 receptors in vascular apoptosis Apoptosis -LRB- programmed cell death -RRB- has been reported to occur in various vascular diseases such as atherosclerosis , restenosis , and hypertension -LSB- 74 , 75 -RSB- . The major cell types undergoing apoptosis in human atherosclerotic lesions are arterial SMC -LSB- 11 , 76 -- 78 -RSB- and macrophages -LSB- 79 -RSB- . In restenosis following balloon angioplasty , there is a peak in the proliferation and apoptosis of rat vascular SMC 14 days postangioplasty -LSB- 76 -RSB- . Furthermore , apoptosis of arterial SMC has been described in animal models of intimal thickenings -LSB- 80 -RSB- and probably takes part in the normal process involved in the control of hyperplasia . In contrast , apoptosis of SMC in advanced human atherosclerotic plaques may destabilize the fibrous lesion to promote plaque rupture and its clinical consequences . As a mediator of cell-to-cell communication , ATP can trigger a variety of biological responses after being rapidly released in large amounts from various sources , including activated platelets , endothelial cells , nerve terminals , antigen-stimulated T cells , and other cell types following hypoxia , stress , and tissue damage . For example , in human umbilical cord vein endothelial cells -LRB- HUVEC -RRB- , a substantial release of ATP -LRB- and UTP -RRB- is induced by shear stress -LSB- 81 -RSB- , which may lead to alterations in the balance between proliferation and apoptosis regulated by P1 adenosine and P2 -LRB- particularly P2X7 -RRB- nucleotide receptors -LSB- 82 -RSB- . P2X7 and P1 receptors have been previously linked to apoptosis in other cell types , including immune cells , astrocytes , and thymocytes -LSB- 83 -- 85 -RSB- . The P2X7R also has been shown to mediate ATP-induced cell death in human embryonic kidney cells -LSB- 86 -RSB- , human cervical epithelial cells -LSB- 87 -RSB- , and primary rat cortical neurons -LSB- 88 -RSB- . In human arterial SMC , adenosine-induced apoptosis is essentially mediated via the A2b-adenosine receptor subtype and involves a cAMP-dependent pathway -LSB- 89 -RSB- . As an important constituent of atherosclerotic plaques , fibroblasts share several features with smooth muscle cells . In human fibroblasts , P2X7 was identified as the main nucleotide receptor involved in the high glucose concentration-dependent responses modulated by ATP , including morphological changes , enhanced apoptosis , caspase-3 activation , and IL-6 release -LSB- 90 -RSB- . In the immune system , ATP also plays important roles through nucleotide receptors in leukocyte functions . P2X7 receptor-mediated apoptosis has been demonstrated in various types of leukocytes , including a lymphocytic cell line , murine thymocytes , murine peritoneal macrophages , human macrophages , mesangial cells , dendritic cells , and microglial cells -LSB- 83 , 91 -- 96 -RSB- . Extracellular ATP acting via the P2X7 receptor activates the transcription factor NFκB by selectively targeting NFκB p65 -LRB- Rel A -RRB- in the N9 mouse microglial cell line -LSB- 97 -RSB- . It also has been reported that the P2X7 receptor modulates macrophage production of TNF-α , IL-1β , and nitric oxide -LRB- NO -RRB- following lipopolysaccharide -LRB- LPS -RRB- exposure -LSB- 98 -RSB- , consistent with a role for the P2X7 receptor in inflammation . In HUVEC , TNF-α markedly increases apoptotic cell death via the activation of caspase-3 -LSB- 74 -RSB- . Recent reports indicate that ATP/ADP activate NFκB and induce apoptosis probably through P2X7 receptors in porcine aortic endothelial cells -LSB- 99 -RSB- . These studies have provided compelling evidence suggesting a role for P2 and P1 receptor-mediated apoptosis in vascular diseases ; however , further studies are needed to determine the precise pathways involved and to accumulate direct evidence that these pathways contribute significantly to the development of atherosclerosis , hypertension , and restenosis . Modulation of P2 receptors in vascular injury Experimental arterial intimal hyperplasia can be induced by balloon angioplasty or by the perivascular placement of a silicone collar around an artery . An influx of leukocytes precedes the migration and proliferation of vascular SMC into the intima in both these models -LSB- 100 -RSB- . In normal adult rat aorta , P2Y2 mRNA was found in the endothelial cell lining , while a sustained expression of P2Y2 mRNA was detected in few medial SMC -LSB- 20 -RSB- . In contrast , P2Y2 mRNA was detected in all medial SMC of rat fetal aortas and in most of the aortic SMC of intimal lesions after balloon angioplasty , with an overexpression in cells lining the lumen both 1 and 3 weeks after injury . In the collar model , neointimal formation appears to be triphasic -LSB- 100 -RSB- . The first phase is characterized by vascular infiltration of leukocytes beginning 2 h after collar placement around a rabbit carotid artery . The second phase begins within 12 h of collar placement and is characterized by medial replication of SMC . The third phase is characterized by the appearance , beginning at day 3 after collar placement , of subendothelial SMC . In situ hybridization with sham-operated rabbit carotid arteries indicated that P2Y2 mRNA expression was localized to CD31-positive aortic endothelial cells and not medial SMC -LSB- 6 -RSB- . High levels of P2Y2 mRNA were detected in medial SMC 3 days after collar placement , before appearance of neointima . At day 14 , all intimal and medial SMC were P2Y2 positive . Fura-2 digital imaging of single SMC , used to measure changes in myoplasmic calcium concentration in response to P2Y receptor agonists , confirmed an increase in P2Y2 receptor activity . However , the same study showed that P2Y4 mRNA was equivalently expressed in sham-operated and collared arteries or cultured rabbit carotid SMC , whereas P2Y6 mRNA was not detected in carotid arteries or cultured SMC . In a more recent study , it was shown that P2Y2 receptor upregulation occurs in stented porcine coronary artery , a clinically relevant model of arterial injury -LSB- 54 -RSB- . P2Y2 receptor mRNA levels were significantly increased in coronary SMC dispersed from stented segments of coronary arteries 3 weeks after stent angioplasty compared with SMC from unstented segments . There was no significant difference observed in levels of P2Y6 mRNA in the stented and unstented artery segments , whereas P2Y4 receptor mRNA was undetectable . Upregulation of functional P2Y receptors also occurs in the basilar artery of the rat double-hemorrhage model -LSB- 7 -RSB- , in the coronary artery of diabetic dyslipidemic pigs -LSB- 8 -RSB- , and in human atherosclerotic lesions -LRB- Seye and Desgranges , unpublished data -RRB- . It has been proposed that upregulation of P2Y receptors could be a potential diagnostic indicator for the early stages of atherosclerosis -LSB- 9 -RSB- . Interestingly , a more recent study showed that high shear stress , associated with vascular diseases , can selectively upregulate P2Y2 and P2Y6 receptors in perfused arterial SMC -LSB- 101 -RSB- . P2X1 and P2X4 receptors have been shown to be upregulated in rabbit intimal thickenings -LSB- 102 -RSB- . Taken together , these findings strongly suggest that at least some P2 receptor subtypes -LRB- most notably P2Y2 -RRB- are implicated in the development of vascular disease . Pathophysiological significance of P2 receptor modulation in vascular injury Migrating and proliferating SMC in the arterial media , together with infiltrating macrophages and T lymphocytes , are the main cell types that comprise atherosclerotic and restenotic lesions -LSB- 1 -RSB- . In the early stages of intimal hyperplasia , SMC are modified from a differentiated , contractile phenotype to an immature , synthetic phenotype , and this enables them to migrate into the intima , to proliferate , and to secrete extracellular matrix components . In many respects , this shift in phenotype is a reversal of the normal differentiation pattern of vascular SMC during fetal and early postnatal life -LSB- 103 , 104 -RSB- . However , there is still a lack of knowledge concerning the phenotypic regulation of SMC during vasculogenesis and vascular disease . A high level of P2 receptor expression could be related to the altered phenotype of SMC in intimal thickenings . Partially dedifferentiated SMC are found in rat arterial intimal lesions after balloon angioplasty -LSB- 105 , 106 -RSB- , and their phenotype has been compared with that of newborn rat aortic SMC -LSB- 107 -RSB- . Interestingly , it has been reported that medial SMC of rat embryonic aorta also exhibit high P2Y2 expression , similar to intimal thickenings -LSB- 20 -RSB- . Other studies have shown that P2Y1 and P2Y2 receptor transcripts are strongly upregulated with phenotypic changes in rat SMC , whereas P2X1 mRNA is completely downregulated , and P2Y4 and P2Y6 mRNA levels are unchanged -LSB- 58 , 28 -RSB- . Taken together , these and other results suggest that P2Y2 receptor expression is upregulated in the entire cell population of intimal thickenings and is closely associated with a poorly differentiated phenotype of SMC . The dramatic increase in P2Y2 mRNA expression observed in balloon angioplasty-induced intimal lesions would suggest increased activity of extracellular nucleotides with consequent enhancement of cell proliferation and vasoreactivity . Indeed , extracellular nucleotides , particularly ATP and UTP , have been shown to induce cell cycle progression and proliferation of cultured arterial SMC -LSB- 21 , 49 , 50 , 51 -RSB- and vasoconstriction in the absence of endothelial cells -LSB- 17 , 108 , 109 -RSB- . Since both neointimal hyperplasia and vasoconstrictive remodeling have been found to be involved in postangioplasty restenosis -LSB- 1 , 5 , 107 , 110 -RSB- , these findings suggest that extracellular nucleotides may play a significant role in this process , at least as long as functional endothelial cells , which regulate intimal thickening -LSB- 111 , 112 -RSB- and nucleotide-induced vasorelaxation -LSB- 113 , 114 -RSB- , are not regenerated . Increased P2Y2 receptor expression in the neointima may by itself be sufficient to enhance the local effects of extracellular nucleotides on the proliferation of SMC . Although the expression of other P2 receptors has been described in arterial SMC -LSB- 21 , 29 , 115 -RSB- , the P2Y2 receptor seems to be more specifically involved in the response of SMC to ATP and UTP -LSB- 116 -RSB- , particularly in the potentiation of proliferation -LSB- 21 , 50 -RSB- . The effects of extracellular nucleotides are not only dependent on the nature and number of P2 receptors present on target cells , but also on the local concentrations of nucleotide agonists . Although in vivo concentrations of extracellular nucleotides are difficult to measure , various in vitro experiments suggest that extracellular nucleotides are released from blood and vascular cells exposed to various physicochemical conditions , e.g. , stress , hypoxia , and other factors -LSB- 117 , 118 , 119 -RSB- associated with the angioplasty process . P2Y2 receptors in SMC are involved in nucleotide-induced constriction of normal arteries -LSB- 17 , 108 , 109 -RSB- . Long lasting alterations in vasomotricity after endothelial cell denudation , resulting in increased sensitivity to vasoconstrictive substances , have previously been demonstrated -LSB- 113 , 114 -RSB- . It appears that like other receptors for vasoconstrictive factors such as angiotensin II -LSB- 120 -RSB- , endothelin -LSB- 121 -RSB- , and platelet-derived growth factor -LRB- PDGF -RRB- -LSB- 122 -RSB- , which are overexpressed in neointima , P2Y2 receptors may play an important role in controlling the vasoactive properties of pathological arteries , particularly in chronic constriction at the lesion site that is postulated to be one of the processes leading to postangioplasty restenosis -LSB- 5 , 110 -RSB- . Conclusion P2 receptor subtypes , including P2Y2 , P2X1 , and P2X4 , appear to play a role in responses to endothelial injury that are thought to be key events in the initiation of atherosclerosis and restenosis after angioplasty . P2Y2R upregulation and activation in endothelial cells and SMC promote leukocyte transmigration and intimal thickening in arteries of animal models of vascular injury , suggesting a possible regulatory role for this receptor in the mechanisms leading to neointimal hyperplasia after angioplasty . Although there are no selective P2Y2 receptor antagonists yet available , recent progress in siRNA technology has made it possible to design small RNA interference molecules that can selectively inhibit P2 receptor subtype expression . Such molecules can be efficiently delivered into the vessel wall using adenoviral vectors . In addition , P2 receptor transgenic mice -LRB- i.e. , mice in which the relevant receptor subtype has been deleted or overexpressed -RRB- will be valuable tools to substantiate the role that nucleotides and P2 receptors play in the etiology of cardiovascular disease . Further delineation of the signaling pathways involved in these P2 receptor-mediated processes may help limit or prevent vascular diseases such as atherosclerosis and restenosis after angioplasty .

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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O B O O O

atherosclerosis
inflammation
migration
nucleotide receptors
proliferation
restenosis
smooth muscle cell