cardiovascular disease ( cvd ) is still the leading cause of mortality worldwide . to date , hypertension is regarded as one of the most important modifiable risk factors of cvd . it has been reported that 54% of stroke cases and 47% of ischemic heart disease cases can be attributed to high blood pressure ( bp).1 , 2 the development of hypertension is a complex process involving many factors.3 although the corresponding interventions have improved hypertension control rates,4 hypertension remains an important public health challenge . unknown risk factors for hypertension need to be studied to improve hypertension prevention and control . ykl40 , also known as human cartilage glycoprotein39 and chitinaselike protein 1 , is a 40  kda heparin and chitinbinding glycoprotein and a member of the mammalian chitinaselike proteins.5 , 6 it is secreted from macrophages , neutrophils , and vascular smooth muscle cells.7 at present , ykl40 has been proposed to be a new marker of inflammation , atherosclerosis , and endothelial dysfunction.8 , 9 in addition , ykl40 was found to increase in individuals with hypertension , especially in those with nondipper hypertension.10 , 11 however , these investigations were crosssectional studies without a clear temporal relationship between ykl40 and hypertension incidence . therefore , whether ykl40 can be considered an independent risk factor for hypertension incidence needs to be elucidated . to address this issue , we conducted a nested casecontrol study in which we measured plasma levels of ykl40 at baseline in a prospective cohort of initially healthy chinese adults who were followed up for hypertension incidence . the present investigation was performed as a nested casecontrol study in 20  343 individuals participating in a populationbased investigation of risk factors for cvds initiated in changshu , jiangsu province of china , from 2007 through 2008 . these individuals lived in rural communities of south china where the economy situation is relatively developed . data on demographic information , lifestyle risk factors , and personal medical history were collected with standard questionnaires by trained staff . alcohol consumption was defined as consuming any type of alcohol beverage at least 12 times during the past 1  year . body weight and height were measured using a regularly calibrated stadiometer and balancebeam scale with participants wearing light clothing and no shoes . body mass index ( bmi ) was calculated as weight in kilograms divided by height in meters squared . participants were advised to avoid alcohol , cigarettes , coffee / tea , and exercise for at least 30  minutes before their bp measurements . three bp measurements were measured using an electronic bp monitor ( omron hem770a , omron healthcare inc , dalian , china ) with a 30second interval . bp was measured with the individual in a sitting position after 5  minutes of rest . hypertension was defined as mean systolic bp 140  mm  hg and/or mean diastolic bp 90  mm  hg or current use of antihypertensive medications . individuals were excluded from the current analysis for the following reasons : baseline hypertension or prevalence or history of coronary heart disease , stroke , chronic kidney diseases , tumors , chronic obstructive pulmonary diseases , or peripheral artery diseases . finally , a total of 12  423 individuals were free of hypertension , cvd , or any other severe disease at baseline . this study was approved by the institutional ethics review board and was conducted in accordance with the guidelines of the declaration of helsinki . data on the personal history of disease and medication during followup were obtained using a standard questionnaire written in chinese and administered by trained staff . participants who reported an incidence of coronary heart diseases , stroke , chronic kidney diseases , tumors , chronic obstructive pulmonary diseases , or peripheral artery diseases during the followup period were asked to provide hospital records , including outpatient , admission records , or discharge summary . experienced physicians carefully reviewed the records to confirm the above diseases and excluded these patients from the study . death data were confirmed after obtaining death certificates from the local citizen registry or the attended hospital . new cases of hypertension were identified in the following ways : ( 1 ) selfreported use of antihypertensive medications in the previous 2  weeks and ( 2 ) systolic bp 140  mm  hg and/or diastolic bp 90  mm  hg during the visit . finally , a total of 1774 new cases of hypertension were ascertained from baseline to the followup end point . for the present study , we selected newonset hypertension patients as cases . as controls , we selected normotensive individuals during the followup throughout the same time interval as the selected cases . among 1774 patients with newonset hypertension , we selected 700 as cases by means of stratified random sampling method according to their resident areas . controls were matched to the cases 1:1 based on age ( birth at the same year ) and sex . if more than one control was available , we would select the person living close to the case as the control . the 700 pairs of cases and controls with complete information and blood samples were finally included in this nested casecontrol study . blood samples were obtained in the morning by venipuncture after a requested overnight fasting period ( at least 8  hours ) and sampled in edta tubes and immediately spun at 3000  rpm for 15  minutes at study entry . plasma samples were stored at 80c and measurements were performed by laboratory technicians who were blinded to the characteristics of the study patients . ykl40 was reported to be stable in blood samples frozen at 80c after several cycles of freezing and thawing.12 plasma ykl40 tests were performed using commercial enzymelinked immunosorbent assay kits ( catalog number dc3l10 ; r&d systems , inc , minneapolis , mn ) according to the manufacturer 's instructions . a standard curve was constructed from which the ykl40 concentrations of unknown samples were determined . intra and interassay coefficients of variation were less than 5% and 7% , respectively . at baseline , categorical variables were presented as number of patients ( percentages ) and compared by unadjusted conditional logistic regression analysis . because of the skewed distribution , the concentration of ykl40 was presented as median with a range between 25th and 75th percentiles . differences of ykl40 levels between the cases and controls were compared using pairedsample wilcoxon signed rank test . the association of natural logtransformed ykl40 with hypertension was assessed using multiple conditional logistic regression analysis adjusting for bmi , smoking , drinking , glucoselowering therapy , lipidlowering therapy , baseline bp , fasting glucose , and lipid levels . in addition , the study population was divided into tertiles on the basis of the distribution of control values . odds ratios ( ors ) and 95% cis were calculated for the upper two categories of ykl40 level with the lowest one as a reference . a test for linear trend was performed by entering categorical variables as continuous parameters in the model . we also set a multiplicative interaction term of ykl40 and age ( > 55 or 55  years ) or sex in the multivariable conditional logistic model to test the interaction effect on hypertension incidence with adjustment for other confounding factors . we assessed whether the addition of ykl40 in the multivariable model made better predictions than a model built only based on conventional risk factors using net reclassification improvement ( nri ) and integrated discrimination improvement ( idi).13 nri represented the incremental ability to accurately reclassify patients with newonset hypertension into higher risk categories and individuals without hypertension into lower ones after the natural logtransformed ykl40 level was incorporated into the prediction models . idi reflected the increase in difference of mean probability to predict hypertension events in cases with that in controls , indicating whether the prediction model with additive natural logtransformed ykl40 level had a better ability to distinguish cases from controls . two conditional logistic regression models were applied to estimate the increased discriminative ability based on the ykl40 , and patients were divided into 3 categories for the risk classification ( < 10% , 10%30% , and > 30% ) . all statistical tests were 2tailed and were significant if the p value was less than 0.05 . statistical analysis was carried out using sas software ( version 9.3 ; sas institute , cary , nc ) . the present investigation was performed as a nested casecontrol study in 20  343 individuals participating in a populationbased investigation of risk factors for cvds initiated in changshu , jiangsu province of china , from 2007 through 2008 . these individuals lived in rural communities of south china where the economy situation is relatively developed . data on demographic information , lifestyle risk factors , and personal medical history were collected with standard questionnaires by trained staff . alcohol consumption was defined as consuming any type of alcohol beverage at least 12 times during the past 1  year . body weight and height were measured using a regularly calibrated stadiometer and balancebeam scale with participants wearing light clothing and no shoes . body mass index ( bmi ) was calculated as weight in kilograms divided by height in meters squared . participants were advised to avoid alcohol , cigarettes , coffee / tea , and exercise for at least 30  minutes before their bp measurements . three bp measurements were measured using an electronic bp monitor ( omron hem770a , omron healthcare inc , dalian , china ) with a 30second interval . bp was measured with the individual in a sitting position after 5  minutes of rest . hypertension was defined as mean systolic bp 140  mm  hg and/or mean diastolic bp 90  mm  hg or current use of antihypertensive medications . individuals were excluded from the current analysis for the following reasons : baseline hypertension or prevalence or history of coronary heart disease , stroke , chronic kidney diseases , tumors , chronic obstructive pulmonary diseases , or peripheral artery diseases . finally , a total of 12  423 individuals were free of hypertension , cvd , or any other severe disease at baseline . this study was approved by the institutional ethics review board and was conducted in accordance with the guidelines of the declaration of helsinki . data on the personal history of disease and medication during followup were obtained using a standard questionnaire written in chinese and administered by trained staff . participants who reported an incidence of coronary heart diseases , stroke , chronic kidney diseases , tumors , chronic obstructive pulmonary diseases , or peripheral artery diseases during the followup period were asked to provide hospital records , including outpatient , admission records , or discharge summary . experienced physicians carefully reviewed the records to confirm the above diseases and excluded these patients from the study . death data were confirmed after obtaining death certificates from the local citizen registry or the attended hospital . new cases of hypertension were identified in the following ways : ( 1 ) selfreported use of antihypertensive medications in the previous 2  weeks and ( 2 ) systolic bp 140  mm  hg and/or diastolic bp 90  mm  hg during the visit . among 12  423 healthy individuals at baseline , 2344 finally , a total of 1774 new cases of hypertension were ascertained from baseline to the followup end point . for the present study , we selected newonset hypertension patients as cases . as controls , we selected normotensive individuals during the followup throughout the same time interval as the selected cases . among 1774 patients with newonset hypertension , we selected 700 as cases by means of stratified random sampling method according to their resident areas . controls were matched to the cases 1:1 based on age ( birth at the same year ) and sex . if more than one control was available , we would select the person living close to the case as the control . the 700 pairs of cases and controls with complete information and blood samples were finally included in this nested casecontrol study . blood samples were obtained in the morning by venipuncture after a requested overnight fasting period ( at least 8  hours ) and sampled in edta tubes and immediately spun at 3000  rpm for 15  minutes at study entry . plasma samples were stored at 80c and measurements were performed by laboratory technicians who were blinded to the characteristics of the study patients . ykl40 was reported to be stable in blood samples frozen at 80c after several cycles of freezing and thawing.12 plasma ykl40 tests were performed using commercial enzymelinked immunosorbent assay kits ( catalog number dc3l10 ; r&d systems , inc , minneapolis , mn ) according to the manufacturer 's instructions . a standard curve was constructed from which the ykl40 concentrations of unknown samples were determined . at baseline , continuous variables were presented as meanssds and compared by pairedsamples t test . categorical variables were presented as number of patients ( percentages ) and compared by unadjusted conditional logistic regression analysis . because of the skewed distribution , the concentration of ykl40 was presented as median with a range between 25th and 75th percentiles . differences of ykl40 levels between the cases and controls were compared using pairedsample wilcoxon signed rank test . the association of natural logtransformed ykl40 with hypertension was assessed using multiple conditional logistic regression analysis adjusting for bmi , smoking , drinking , glucoselowering therapy , lipidlowering therapy , baseline bp , fasting glucose , and lipid levels . in addition , the study population was divided into tertiles on the basis of the distribution of control values . odds ratios ( ors ) and 95% cis were calculated for the upper two categories of ykl40 level with the lowest one as a reference . a test for linear trend was performed by entering categorical variables as continuous parameters in the model . we also set a multiplicative interaction term of ykl40 and age ( > 55 or 55  years ) or sex in the multivariable conditional logistic model to test the interaction effect on hypertension incidence with adjustment for other confounding factors . we assessed whether the addition of ykl40 in the multivariable model made better predictions than a model built only based on conventional risk factors using net reclassification improvement ( nri ) and integrated discrimination improvement ( idi).13 nri represented the incremental ability to accurately reclassify patients with newonset hypertension into higher risk categories and individuals without hypertension into lower ones after the natural logtransformed ykl40 level was incorporated into the prediction models . idi reflected the increase in difference of mean probability to predict hypertension events in cases with that in controls , indicating whether the prediction model with additive natural logtransformed ykl40 level had a better ability to distinguish cases from controls . two conditional logistic regression models were applied to estimate the increased discriminative ability based on the ykl40 , and patients were divided into 3 categories for the risk classification ( < 10% , 10%30% , and > 30% ) . all statistical tests were 2tailed and were significant if the p value was less than 0.05 . statistical analysis was carried out using sas software ( version 9.3 ; sas institute , cary , nc ) . in table  1 , case and control patients were matched for age ( 52.6  years on average ) and sex ( 33.3% male ) . as expected , initially healthy persons who subsequently developed hypertension ( cases ) were more likely to be drinkers ; have higher bmi , triglyceride , and bp levels ; and lower hdl cholesterol levels compared with those who remained free of hypertension ( controls ) at baseline . among the total population and most subgroups however , hypertension patients had a significantly higher level of ykl40 ( 69.1 [ 30127.2 ] ng / ml ) than controls ( 55.4 [ 22.4111.1 ] ng / ml ) in the male subgroup ( table  2 ) . baseline characteristics according to 700 hypertension patients and 700 age and sexmatched controls bmi indicates body mass index . comparison of ykl40 levels between patients and controls among the total population and different subgroups as shown in table  3 , ykl40 was not associated with hypertension incidence among total patients . in the male subgroup , there was a significant association between perunit increase of logtransformed ykl40 and hypertension incidence ( or , 1.24 ; p=0.04 ) . in the multivariate analysis , patients in the third tertile had a 1.89fold increased risk of hypertension compared with those in the lowest tertile ( p<0.05 ) . ors of hypertension positively increased with ykl40 levels ( p values for linear trend=0.02 ) . after further adjusting for baseline bp , men in the third tertile still had a 2.09fold increased risk of hypertension compared with those in the lowest tertile ( p<0.05 ) . ors of hypertension also positively and significantly increased with ykl40 levels ( p for linear trend=0.02 ) . in the subgroup of patients older than 55 years , compared with the patients in the lowest tertile , those in the third tertile had a 1.59 increased risk of hypertension with a significant linear trend . in addition , an interaction effect was observed between ykl40 and sex ( p  for interaction < 0.01 ) , not between ykl40 and age ( p for interaction=0.21 ) . multivariable adjusted odds ratios ( 95% cis ) of hypertension for upper tertiles of plasma ykl40 levels among the total population and different subgroups model 1 : adjusted for body mass index , drinking status , smoking , glucoselowering therapy , lipidlowering therapy , blood glucose , total cholesterol , triglycerides , and highdensity lipoprotein cholesterol . model 2 : model 1 plus adjustment for baseline systolic and diastolic blood pressure . with respect to the ability of the prediction model to distinguish hypertension cases from controls , we found that the inclusion of ykl40 as a predictor conferred discernible improvement in discriminatory performance on models both in the age older than 55  years and male subgroups ( table  4 ) . among male patients , with those stratified into hypertension risk categories according to the predicted risk probabilities , we found that the prediction model with ykl40 had a greater ability not only to reclassify hypertension cases into higher risk categories , but to reclassify controls into lower risk categories , relative to the model without ykl40 ( nri , 41.2% ; 95% ci , 23.47%58.93% ; p<0.01 ) . also , the increased performance was further corroborated by the significant improvement in the capability to accurately identify hypertension events in cases rather than in controls after ykl40 was incorporated into the prediction model ( idi , 0.73% ; 95% ci , 0.06%1.4% ; p=0.03 ) . comparison regarding discrimination performance between multivariateadjusted models with and without taking ykl40 into consideration among the total population and different subgroups conventional factors included body mass index , drinking status , smoking , glucoselowering therapy , lipidlowering therapy , blood glucose , total cholesterol , triglyceride , highdensity lipoprotein cholesterol , and systolic and diastolic blood pressure . in this study , we found that high levels of ykl40 at baseline predicted hypertension incidence in men . this predictive property is independent of baseline conventional cvd risk factors and improves discrimination between cases and controls beyond these factors . endothelial dysfunction and inflammation are two key steps in the pathophysiology of hypertension.14 ykl40 , which is produced by macrophages and neutrophils within inflamed tissues , is associated with atherosclerosis and promotes vascular smooth muscle cells attachment , spreading , and migration.8 previous studies have detected an association between ykl40 level and bp . ma et  al10 reported that patients with either nonmicroalbuminuric ( 61.63  ng / ml ) or microalbuminuric ( 98.78  ng / ml ) hypertension had a significantly higher level of ykl40 compared with healthy controls ( 37.85  ng / ml ) . bakirci et  al11 further found that ykl40 level elevation was more obvious among patients with nondipper hypertension . ykl40 was a good predictor of nondipper hypertension pattern with an area of 0.774 under the receiver operating characteristic curve . however , it is difficult to determine whether the elevation of ykl40 preceded or was a result of bp rising because of the nonprospective study design used in these studies . in addition , the sample sizes of the above two studies were less than 200 and the statistical power was relatively low . whether the detected association between ykl40 and bp could be applied to the total population or certain prespecified subgroup remains to be further studied . in contrast , our present study is a large sample nested casecontrol study with high statistical power . the negative association between ykl40 and hypertension incidence among the total population and the positive association among male patients seems more plausible . in this study , although there was no difference inykl40 levels between the men and the women , the effect of ykl40 on hypertension development progress is indeed different . as a novel marker of endothelial dysfunction , we speculated that its function was tightly associated with endothelial function of the individuals themselves . flowmediated dilation ( fmd ) is often used as a noninvasive assessment of endothelial function.15 skaug et  al16 evaluated the endothelial function by fmd in 4739 healthy adults . among them , men had a significantly lower fmd than women ( 4.33.9% versus 5.34.5% , p<0.01 ) . lower fmd values in men probably indicate poor endothelial function and substantial prevalence of subclinical atherosclerosis . the sex differences in risk for atherosclerotic events have previously been demonstrated by other studies.17 , 18 , 19 it has been suggested that sex hormones conserve fmd in women and estrogen played a protective role in endothelial function.20 , 21 in contrast , cigarette smoking aggravates inflammation and endothelial dysfunction in men.22 , 23 the significant interaction between ykl40 and sex in our study further suggests that the more undesirable endothelial dysfunction in men promoted the ykl40 effect on hypertension incidence . our study used a prospective nested casecontrol design in which plasma samples for all study participants were obtained at study entry , a long time interval before the occurrence of the hypertension . such a study design considerably reduces potential biases inherent in crosssectional or retrospective studies in which plasma sampling is performed after rather than before the onset of disease . in addition , by matching case and control participants by age and sex and adjusting for additional factors such as blood glucose and lipid levels in multivariable analysis , the present study had a high degree of comparability between cases and controls and controlled for some important confounding factors in the analysis . first , the nested casecontrol design of the study may overestimate the true predictive capacity of the models because of the relative high event rate in casecontrol studies.24 second , all participants were followed up only one time for newonset hypertension . therefore , it is difficult for us to estimate the accurate time of onset of new hypertension cases . furthermore , baseline ykl40 levels in our study were determined with a single measurement so that the exposure misclassification bias is inevitable but probably low , because a previous study has proved plasma ykl40 stability over time.12 finally , it is unavoidable that there is selection bias because of the nested casecontrol design . because of the number of subgroup analyses performed , the statistical significance may have occurred by chance alone . this study has several strengths that deserve mention . our study used a prospective nested casecontrol design in which plasma samples for all study participants were obtained at study entry , a long time interval before the occurrence of the hypertension . such a study design considerably reduces potential biases inherent in crosssectional or retrospective studies in which plasma sampling is performed after rather than before the onset of disease . in addition , by matching case and control participants by age and sex and adjusting for additional factors such as blood glucose and lipid levels in multivariable analysis , the present study had a high degree of comparability between cases and controls and controlled for some important confounding factors in the analysis . however , there are also some limitations that should be mentioned . first , the nested casecontrol design of the study may overestimate the true predictive capacity of the models because of the relative high event rate in casecontrol studies.24 second , all participants were followed up only one time for newonset hypertension . therefore , it is difficult for us to estimate the accurate time of onset of new hypertension cases . furthermore , baseline ykl40 levels in our study were determined with a single measurement so that the exposure misclassification bias is inevitable but probably low , because a previous study has proved plasma ykl40 stability over time.12 finally , it is unavoidable that there is selection bias because of the nested casecontrol design . because of the number of subgroup analyses performed , the statistical significance may have occurred by chance alone . controlling ykl40 at a relative low level may be beneficial for hypertension prevention in the male population . this study was supported by the national natural science foundation of china ( grant no .
backgroundhuman cartilage glycoprotein39 ( ykl40 ) has been suggested to be a new marker of inflammation , atherosclerosis , and endothelial dysfunction . however , whether a higher level of ykl40 is an independent risk factor for hypertension incidence is still unknown.methods and resultsin a nested casecontrol study within a prospective cohort of 12  423 initially healthy chinese adults , we measured baseline plasma concentrations of ykl40 among 700 newonset hypertension cases and 700 age and sexmatched controls . 
 multiple conditional logistic regression analyses were used to calculate the odds ratios ( 95% cis ) of hypertension associated with higher levels of ykl40 both in the total population and in the age ( > 55 and 55 years ) and sexmatched subgroups . among the total population , 
 ykl40 levels were not associated with hypertension risk . in the subgroup older than 55  years , odds ratios ( 95% cis ) of hypertension for those in the two higher tertiles of ykl40 were 1.23 ( 0.771.97 ) and 1.59 ( 0.992.55 ) ( p for linear trend=0.05 ) . in the male subgroup , 
 odds ratios ( 95% cis ) of hypertension for those in the two higher tertiles of ykl40 were 1.55 ( 0.882.72 ) and 2.09 ( 1.143.82 ) ( p for linear trend=0.02 ) . 
 an interaction effect was observed between ykl40 and sex ( p for interaction < 0.01 ) but not between ykl40 and age ( p for interaction=0.21 ) . 
 high ykl40 level significantly increased hypertension risk in men but decreased hypertension risk with a trend although not significant in women.conclusionsthis study suggests that ykl40 is associated with hypertension incidence only among men . 
 the study findings need to be further verified by prospective cohort studies or clinical trials .