heart failure ( hf ) can result from cardiac overload or injury as well as from a complex interplay of genetic , neurohormonal , and inflammatory factors . hf is classified as being associated with a reduced ejection fraction ( hfref ) or with a normal ejection fraction ( hfnef ) . the percentages of patients and the mortality rates are similar in patients with hfref and hfnef . the likelihood of survival is higher in patients with hfref than in those with hfnef , and the survival rate in hfnef has not significantly improved in recent years . various biomarkers , including those for inflammation and neurohormones , provide important information about the pathogenesis , risk stratification , and diagnosis of hf and are used to monitor response to therapy . inflammatory markers are closely associated with pathogenesis , poor functional state , and adverse prognosis in patients with hf . pentraxin 3 ( ptx3 ) is a newly identified member of the pentraxin superfamily , which includes creactive protein ( crp ) and serum amyloid p. in contrast to crp , which is produced by the liver , ptx3 is produced by various cell types in various tissues , especially in the vasculature , in response to inflammatory stimuli . ptx3 may reflect local inflammatory status in tissues and thus may be a new biomarker of inflammation . we recently demonstrated that plasma levels of ptx3 , but not highsensitivity crp ( hscrp ) , were significantly higher in patients with hfnef than in nonhf patients . in addition , these elevated levels of ptx3 were significantly and independently correlated with the presence of hfnef in patients with normal left ventricular ejection fraction ( lvef ) . other studies have also shown that ptx3 is a predictor of adverse clinical outcome in patients with hfref . although much attention has been focused on hfref , less is known regarding factors that correlate with clinical outcome in patients with hfnef . the irbesartan in heart failure with preserved ejection fraction ( ipreserve ) study demonstrated that age , presence of diabetes mellitus , history of hospitalization for hf , new york heart association classification , nterminal probtype natriuretic peptide levels , and lvef were associated with adverse outcomes in patients with hfnef . this study was designed to determine the ability of plasma ptx3 levels to predict future cardiovascular events in patients with hfnef . we initially screened 747 chronic hf patients under a clinically stable condition with new york heart association functional class ii to iv who were referred to kumamoto university hospital , kumamoto , japan . none of the patients had any noncardiac causes of hflike symptoms , especially lung disease ( eg , chronic obstructive lung disease ) . the diagnosis of hfnef was based on the criteria formulated by the european working group on hfnef . hfref was defined as reduced systolic left ventricular ( lv ) function ( lvef50% ) . hfnef was defined as normal or mildly abnormal systolic lv function ( lvef>50% ) with lv diastolic dysfunction . lv diastolic dysfunction for hfnef was defined as ( 1 ) a ratio of mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ( e / e ) 15 ; ( 2 ) e / e 8 and < 15 and btype natriuretic peptide ( bnp ) levels > 200 pg / ml ; or ( 3 ) e / e 8 and < 15 and lv mass index ( lvmi ) 122 g / m ( females ) or 149 g / m ( males ) . patients were excluded if they had active systemic inflammatory disease , chronic renal failure requiring hemodialysis , collagen disease , presence of malignancy , or acute coronary syndrome within 3 months preceding enrollment . the study complied with the declaration of helsinki regarding investigation in humans and was approved by the institutional review committee of each institution . the study was also conducted in accordance with the guidelines of the ethics committee of our institution . this study was registered at the university hospital medical information network clinical trials registry with the identification number umin000002170 . flow chart showing the protocol that was used for enrollment of hfnef patients in the present study . bnp indicates btype natriuretic peptide ; e / e , ratio of mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; hf , heart failure ; hfnef , hf with normal ejection fraction ; lvef , left ventricular ejection fraction ; lvmi , left ventricular mass index ; nyha , new york heart association . ptx3 levels were measured using a highsensitivity enzymelinked immunosorbent assay system ( values ranged from 0.1 to 20 ng / ml ; perseus proteomics ) . other factors that were measured included serum hscrp levels and plasma tumor necrosis factor , interleukin6 , and bnp levels . echocardiography was performed in standard parasternal and apical views by a specialized echocardiologist using commercially available ultrasound systems ( vivid 7 , gevingmed ultrasound ; aplio xg , toshiba ) . lvef was measured in biplane apical ( 2 and 4chamber ) views using a modification of simpson 's method . the e / e ratio is used as an index of lv filling pressure and abnormal lv relaxation . to assess lv diastolic function , e / e was measured , as described previously , as was the medial annulus of e / e. lv mass was calculated , as described previously , and the lvmi was expressed relative to body surface area . patients with hfnef were followed prospectively at the outpatient clinic until december 2012 or until an endpoint occurred . the endpoint was a composite of cardiovascular death , nonfatal mi , unstable angina pectoris , nonfatal ischemic stroke , hospitalization for hf decompensation , or coronary revascularization . cardiovascular events were ascertained from a review of medical records and confirmed by direct contact with the patients , their families , and physicians . patients were divided into low and highptx3 groups based on the median concentration of ptx3 ( 3.0 ng / ml ) . cardiovascular death was defined as death because of mi ( within 28 days ) , congestive heart failure , or documented sudden death without apparent noncardiovascular causes . mi was diagnosed as a rise or fall in cardiac biomarkers ( plasma creatine kinasemb or cardiac troponint ) greater than the 99th percentile of the upper limit of normal , together with evidence of myocardial ischemia with at least one of the following : ecg changes ( recent stt changes , left bundle branch block , or a pathological q wave ) or imaging evidence of new loss of viable myocardium or new regional wall motion abnormality . unstable angina pectoris was diagnosed from new or accelerating symptoms of myocardial ischemia accompanied by recent ischemic sttwave changes . ischemic stroke was diagnosed by both neurological symptoms and radiological evidence , excluding intracranial hemorrhage . hospitalization for hf decompensation was defined as admission for symptoms typical of hf with objective signs of worsening hf requiring intravenous drug administration . coronary revascularization was defined as coronary artery bypass grafting or percutaneous coronary intervention in patients with stable angina pectoris . for subjects experiencing 2 or more cardiovascular events , parameters with a normal distribution , as assessed by the shapirowilk test , are expressed as meansd . parameters with a skewed distribution , such as ptx3 , hscrp , tumor necrosis factor , interleukin6 , and bnp levels , as well as e / e and lvmi , are shown as median values ( interquartile range ) and were logarithmically transformed before linear regression analysis . differences between continuous variables were analyzed by the unpaired t test and the mannwhitney u test , as appropriate . linear regression analysis was used to determine the associations between ptx3 and bnp levels , lvmi , inflammatory makers , and cardiovascular events . survival data were analyzed by the kaplanmeier method and assessed by the logrank test . age and the ability of any marker to predict cardiovascular events was assessed by cox proportional hazards regression analysis . in cox proportional hazards regression analyses , associations between groups and all other parameters were first analyzed by univariate analysis followed by multivariable analysis after adjustment for factors that were significant with univariate analysis . model 1 incorporated the inflammatory makers ptx3 , hscrp , tumor necrosis factor , interleukin6 , and bnp . model 2 incorporated the 5 prognostic factors ( pf5 ) that were identified during the ipreserve study in patients with hfnef  age , presence of diabetes mellitus , previous hospitalization for hf , new york heart association classification , and lvef  as well as bnp and ptx3 . model 4 incorporated pf5 , bnp , hscrp , and ptx3 . estimates of the cstatistic for cox proportional hazards regression models were calculated . calibration of cox regression models was also performed by the grnnesby and borgan calibration test . the incremental effects of addition of ptx3 to pf5 and bnp levels to predict future cardiovascular events were evaluated using the net classification index ( nri ) , as previously described . patients were stratified into 1 of 3 risk categories based on pf5 and bnp levels measured during the mean 30month followup period : low risk ( 0% to < 10% ) , intermediate risk ( 10% to 20% ) , or high risk ( > 20% ) . ptx3 was subsequently used to reclassify the risk category for ascertaining whether there would be improvement in the nri . the nri was calculated using the following equation : nri=([number of events reclassified as highernumber of events reclassified as lower]/number of events)([number of nonevents reclassified as lowernumber of nonevents reclassified as higher]/number of nonevents ) . all analyses were performed using spss version 19.0j for windows ( ibm corporation ) , stata version 11 ( stata corporation ) , and sas version 9.1.3 ( sas institute inc ) . we initially screened 747 chronic hf patients under a clinically stable condition with new york heart association functional class ii to iv who were referred to kumamoto university hospital , kumamoto , japan . none of the patients had any noncardiac causes of hflike symptoms , especially lung disease ( eg , chronic obstructive lung disease ) . the diagnosis of hfnef was based on the criteria formulated by the european working group on hfnef . hfref was defined as reduced systolic left ventricular ( lv ) function ( lvef50% ) . hfnef was defined as normal or mildly abnormal systolic lv function ( lvef>50% ) with lv diastolic dysfunction . lv diastolic dysfunction for hfnef was defined as ( 1 ) a ratio of mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ( e / e ) 15 ; ( 2 ) e / e 8 and < 15 and btype natriuretic peptide ( bnp ) levels > 200 pg / ml ; or ( 3 ) e / e 8 and < 15 and lv mass index ( lvmi ) 122 g / m ( females ) or 149 g / m ( males ) . patients were excluded if they had active systemic inflammatory disease , chronic renal failure requiring hemodialysis , collagen disease , presence of malignancy , or acute coronary syndrome within 3 months preceding enrollment . the study complied with the declaration of helsinki regarding investigation in humans and was approved by the institutional review committee of each institution . the study was also conducted in accordance with the guidelines of the ethics committee of our institution . this study was registered at the university hospital medical information network clinical trials registry with the identification number umin000002170 . flow chart showing the protocol that was used for enrollment of hfnef patients in the present study . bnp indicates btype natriuretic peptide ; e / e , ratio of mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; hf , heart failure ; hfnef , hf with normal ejection fraction ; lvef , left ventricular ejection fraction ; lvmi , left ventricular mass index ; nyha , new york heart association . ptx3 levels were measured using a highsensitivity enzymelinked immunosorbent assay system ( values ranged from 0.1 to 20 ng / ml ; perseus proteomics ) . other factors that were measured included serum hscrp levels and plasma tumor necrosis factor , interleukin6 , and bnp levels . echocardiography was performed in standard parasternal and apical views by a specialized echocardiologist using commercially available ultrasound systems ( vivid 7 , gevingmed ultrasound ; aplio xg , toshiba ) . lvef was measured in biplane apical ( 2 and 4chamber ) views using a modification of simpson 's method . the e / e ratio is used as an index of lv filling pressure and abnormal lv relaxation . to assess lv diastolic function , e / e was measured , as described previously , as was the medial annulus of e / e. lv mass was calculated , as described previously , and the lvmi was expressed relative to body surface area . patients with hfnef were followed prospectively at the outpatient clinic until december 2012 or until an endpoint occurred . the endpoint was a composite of cardiovascular death , nonfatal mi , unstable angina pectoris , nonfatal ischemic stroke , hospitalization for hf decompensation , or coronary revascularization . cardiovascular events were ascertained from a review of medical records and confirmed by direct contact with the patients , their families , and physicians . patients were divided into low and highptx3 groups based on the median concentration of ptx3 ( 3.0 ng / ml ) . cardiovascular death was defined as death because of mi ( within 28 days ) , congestive heart failure , or documented sudden death without apparent noncardiovascular causes . mi was diagnosed as a rise or fall in cardiac biomarkers ( plasma creatine kinasemb or cardiac troponint ) greater than the 99th percentile of the upper limit of normal , together with evidence of myocardial ischemia with at least one of the following : ecg changes ( recent stt changes , left bundle branch block , or a pathological q wave ) or imaging evidence of new loss of viable myocardium or new regional wall motion abnormality . unstable angina pectoris was diagnosed from new or accelerating symptoms of myocardial ischemia accompanied by recent ischemic sttwave changes . ischemic stroke was diagnosed by both neurological symptoms and radiological evidence , excluding intracranial hemorrhage . hospitalization for hf decompensation was defined as admission for symptoms typical of hf with objective signs of worsening hf requiring intravenous drug administration . coronary revascularization was defined as coronary artery bypass grafting or percutaneous coronary intervention in patients with stable angina pectoris . for subjects experiencing 2 or more cardiovascular events , parameters with a normal distribution , as assessed by the shapirowilk test , are expressed as meansd . parameters with a skewed distribution , such as ptx3 , hscrp , tumor necrosis factor , interleukin6 , and bnp levels , as well as e / e and lvmi , are shown as median values ( interquartile range ) and were logarithmically transformed before linear regression analysis . differences between continuous variables were analyzed by the unpaired t test and the mannwhitney u test , as appropriate . linear regression analysis was used to determine the associations between ptx3 and bnp levels , lvmi , inflammatory makers , and cardiovascular events . the ability of any marker to predict cardiovascular events was assessed by cox proportional hazards regression analysis . in cox proportional hazards regression analyses , associations between groups and all other parameters were first analyzed by univariate analysis followed by multivariable analysis after adjustment for factors that were significant with univariate analysis . model 1 incorporated the inflammatory makers ptx3 , hscrp , tumor necrosis factor , interleukin6 , and bnp . model 2 incorporated the 5 prognostic factors ( pf5 ) that were identified during the ipreserve study in patients with hfnef  age , presence of diabetes mellitus , previous hospitalization for hf , new york heart association classification , and lvef  as well as bnp and ptx3 . calibration of cox regression models was also performed by the grnnesby and borgan calibration test . the incremental effects of addition of ptx3 to pf5 and bnp levels to predict future cardiovascular events were evaluated using the net classification index ( nri ) , as previously described . patients were stratified into 1 of 3 risk categories based on pf5 and bnp levels measured during the mean 30month followup period : low risk ( 0% to < 10% ) , intermediate risk ( 10% to 20% ) , or high risk ( > 20% ) . ptx3 was subsequently used to reclassify the risk category for ascertaining whether there would be improvement in the nri . the nri was calculated using the following equation : nri=([number of events reclassified as highernumber of events reclassified as lower]/number of events)([number of nonevents reclassified as lowernumber of nonevents reclassified as higher]/number of nonevents ) . all analyses were performed using spss version 19.0j for windows ( ibm corporation ) , stata version 11 ( stata corporation ) , and sas version 9.1.3 ( sas institute inc ) . table 1 shows the clinical characteristics of the participating patients . the median ptx3 level was 3.0 ng / ml . mean age , new york heart association class , and bnp levels were higher and mean body mass index , waist circumference and estimated glomerular filtration rate were lower in the highptx3 group ( > 3.0 ng / ml ) than in the lowptx3 group ( 3.0 ng / ml ) . lvmi values were significantly higher in the highptx3 group than in the lowptx3 group , whereas lvef and e / e were similar in these 2 groups . simple linear regression analysis showed a positive and significant correlation between ln(ptx3 ) and ln(bnp ) ( r=0.295 , p<0.001 ) ( figure 2a ) and a significant but weak positive correlation between ln(ptx3 ) and ln(lvmi ) ( r=0.114 , p<0.05 ) ( figure 2b ) . furthermore , simple linear regression analysis showed a significant but weak positive correlation between ln(ptx3 ) and ln(tumor necrosis factor ) ( r=0.106 , p<0.05 ) but not ln(hscrp ) ( p=0.11 ) or ln(interleukin6 ) ( p=0.80 ) . demographic and clinical characteristics of patients with hfnef data are meansd , number of patients ( % ) , or median ( interquartile range ) . acei indicates angiotensinconverting enzyme inhibitors ; arb , angiotensin ii receptor blocker ; bnp , btype natriuretic peptide ; cad , coronary artery disease ; e / e , mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; gfr , glomerular filtration rate ; hdl , highdensity lipoprotein ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hscrp , highsensitive creactive protein ; il6 , interleukin6 ; ldl , lowdensity lipoprotein ; lv , left ventricle ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. relationships between ptx3 and bnp levels and ptx3 levels and lvmi . simple linear regression analysis shows a positive and significant correlation between ( a ) ln(ptx3 ) and ln(bnp ) ( r=0.295 , p<0.001 ) and ( b ) a significant but weak positive correlation between ln(ptx3 ) and ln(lvmi ) ( r=0.114 , p<0.05 ) . bnp indicates btype natriuretic peptide ; lvmi ; left ventricular mass index ; ptx3 , pentraxin 3 . the mean followup period was 30 months ( range : 1 to 59 months ) . during this time , 106 of these patients experienced cardiovascular events , including cardiovascular death ( n=9 ) , nonfatal mi ( n=4 ) , unstable angina pectoris ( n=13 ) , ischemic stroke ( n=9 ) , hospitalization for hf decompensation ( n=41 ) , and coronary revascularization ( n=30 ) . the frequency of cardiovascular events was significantly higher in the highptx3 group ( n=75 ) than in the lowptx3 group ( n=31 , p<0.001 ) ( table 2 ) . hfrelated cardiovascular events , including cardiovascular death and hospitalization for hf decompensation , were significantly higher in the highptx3 group ( n=41 ) than in the lowptx3 group ( n=9 ) ( p<0.001 ) ( table 2 ) . kaplanmeier analysis showed that the probability of cardiovascular events was also significantly higher in the highptx3 group than in the lowptx3 group ( p<0.001 , logrank test ) ( figure 3a ) . cardiovascular events in hfnef patients with low and high plasma ptx3 levels differences between the groups were assessed by the logrank test . hf indicates heart failure ; hfrelated cardiovascular events , cardiovascular death and hospitalization for hf decompensation ; hfnef , heart failure with normal left ventricular ejection fraction ; ptx3 , pentraxin 3 . kaplanmeier analysis for the probability of cardiovascular events . patients with ( a ) high ptx3 levels and low ptx3 levels , ( b ) subgroups of patients with high and low ptx3 levels plus bnp , and ( c ) subgroups of new york heart association class ii patients with high and low ptx3 levels are shown . bnp indicates btype natriuretic peptide ; ptx3 , pentraxin 3 . the combination of ptx3 and bnp levels led to identification of subgroups ( n=116 , highptx3 and bnp group ; n=64 , highptx3 and lowbnp group ; n=64 , lowptx3 and highbnp group ; and n=116 , lowptx3 and bnp group ) with significantly different probabilities of cardiovascular events ( p<0.001 , logrank test ) ( figure 3b ) . in the lowbnp ( p=0.02 ) and highbnp ( p<0.01 ) groups , the probability of cardiovascular events was significantly higher in those with high ptx3 levels than in those with low ptx3 levels . even among patients with mild hf symptoms ( new york heart association class ii ) , the outcomes were poorer in patients with high ptx3 levels than in those with low ptx3 levels ( p<0.001 , logrank test ) ( figure 3c ) . the results of univariate and multivariable cox proportional hazards analyses for cardiovascular events are shown in tables 3 and 4 . multivariable cox proportional hazards analysis identified ptx3 ( hazard ratio [ hr ] : 1.16 ; 95% ci : 1.05 to 1.27 ; p<0.01 ) , highdensity lipoprotein cholesterol ( hr : 0.99 ; 95% ci : 0.97 to 0.99 ; p<0.05 ) , lvmi ( hr : 1.03 ; 95% ci : 1.05 to 1.27 ) , and bnp ( hr : 1.08 ; 95% ci : 1.03 to 1.14 ; p<0.001 ) as predictors of future cardiovascular events after adjustment for significant factors that were identified by univariate analysis ( table 3 ) . using the forced inclusion model , which evaluated levels of inflammatory markers and bnp ( model 1 ) in multivariable cox hazards analysis , ptx3 , but not hscrp , significantly predicted cardiovascular events ( hr : 1.17 ; 95% ci : 1.07 to 1.28 ; p<0.001 ) . in the forced inclusion model , which included pf5 and bnp , ptx3 significantly predicted cardiovascular events ( model 2 : hr : 1.16 ; 95% ci : 1.05 to 1.27 ; p<0.01 ; model 4 : hr : 1.16 ; 95% ci : 1.06 to 1.27 ; p<0.01 ) ( table 4 ) . furthermore , multivariable cox proportional hazards analysis identified ptx3 ( hr : 1.16 ; 95% ci : 1.01 to 1.35 ; p<0.05 ) and bnp ( hr : 1.21 ; 95% ci : 1.11 to 1.31 ; p<0.001 ) as predictors of future hfrelated cardiovascular events , including cardiovascular death and hospitalization for hf decompensation , after adjustment for significant factors identified by univariate analysis . we classified patients with hfnef into 4 groups according to the levels of ln(ptx3 ) : first quartile ( < 0.59 , ptx3 : < 1.80 ng / ml ) , second quartile ( 0.59 to 1.10 , ptx3 : 1.80 to 3.00 ng / ml ) , third quartile ( 1.10 to 1.35 , ptx3 : 3.00 to 3.85 ng / ml ) , and fourth quartile ( > 1.35 , ptx3 : > 3.85 ng / ml ) ; the relative risk of total cardiovascular events was 1 ( reference ) , 1.36 , 2.71 ( p<0.001 versus the reference ) , and 3.17 ( p<0.001 versus the reference ) , respectively . the logtransformation values of ptx3 demonstrated a linear association with the occurrence of cardiovascular events in patients with hfnef ( r=0.965 , p=0.03 ) . cox proportional hazards analysis of factors that were predictive of future cardiovascular events in patients with hfnef after adjustment for significant factors identified by univariate analysis bnp indicates btype natriuretic peptide ; cad , coronary artery disease ; e / e , mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; gfr , glomerular filtration rate ; hdl , highdensity lipoprotein ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hr , hazard ratio ; hscrp , highsensitivity creactive protein ; il6 , interleukin6 ; ldl , lowdensity lipoprotein ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. cox proportional hazards analysis of factors that were associated with future cardiovascular events in hfnef patients using forced inclusion models bnp indicates btype natriuretic peptide ; ci , confidence interval ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hr , hazard ratio ; hscrp , highsensitive creactive protein ; il6 , interleukin6 ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. we also estimated the cstatistics of pf5 alone . separate incorporation of ptx3 and bnp into pf5 increased the cstatistics for the prediction of future cardiovascular events from 0.617 for pf5 alone to 0.656 for pf5 plus ptx3 and pf5 plus bnp ( table 5 ) . moreover , incorporation of ptx3 and bnp into pf5 increased the cstatistics to 0.683 ( p<0.05 ) . in addition , the pf5 plus bnp plus ptx3 model performed better than the pf5 plus bnp model ( cstatistics : 0.683 versus 0.656 ) . we also confirmed good calibration for the analysis by grnnesby and borgan statistics ( p=0.19 ) . cstatistics for cox proportional hazards analysis predicting future cardiovascular events in patients with heart failure with normal ejection fraction bnp indicates btype natriuretic peptide ; pf5 , the 5 factors identified in the ipreserve study as prognostic in patients with heart failure with normal ejection fraction ( age , presence of diabetes mellitus , previous hospitalization for heart failure , new york heart association classification , and left ventricular ejection fraction ) ; ptx3 , pentraxin 3 . the nri was significant after ptx3 was included : 12.2% for patients without cardiovascular events , 0% for those with cardiovascular events , and 12.2% overall ( p<0.01 ) ( table 6 ) . reclassification of the risk of pf5 and bnp for cardiovascular events after the addition of ptx3 bnp indicates btype natriuretic peptide ; pf5 , the 5 factors identified in the ipreserve study as prognostic in patients with heart failure with normal ejection fraction ( age , presence of diabetes mellitus , previous hospitalization for heart failure , new york heart association classification , and left ventricular ejection fraction ) ; ptx3 , pentraxin 3 . table 1 shows the clinical characteristics of the participating patients . the median ptx3 level was 3.0 ng / ml . mean age , new york heart association class , and bnp levels were higher and mean body mass index , waist circumference and estimated glomerular filtration rate were lower in the highptx3 group ( > 3.0 ng / ml ) than in the lowptx3 group ( 3.0 ng / ml ) . lvmi values were significantly higher in the highptx3 group than in the lowptx3 group , whereas lvef and e / e were similar in these 2 groups . simple linear regression analysis showed a positive and significant correlation between ln(ptx3 ) and ln(bnp ) ( r=0.295 , p<0.001 ) ( figure 2a ) and a significant but weak positive correlation between ln(ptx3 ) and ln(lvmi ) ( r=0.114 , p<0.05 ) ( figure 2b ) . furthermore , simple linear regression analysis showed a significant but weak positive correlation between ln(ptx3 ) and ln(tumor necrosis factor ) ( r=0.106 , p<0.05 ) but not ln(hscrp ) ( p=0.11 ) or ln(interleukin6 ) ( p=0.80 ) . demographic and clinical characteristics of patients with hfnef data are meansd , number of patients ( % ) , or median ( interquartile range ) . acei indicates angiotensinconverting enzyme inhibitors ; arb , angiotensin ii receptor blocker ; bnp , btype natriuretic peptide ; cad , coronary artery disease ; e / e , mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; gfr , glomerular filtration rate ; hdl , highdensity lipoprotein ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hscrp , highsensitive creactive protein ; il6 , interleukin6 ; ldl , lowdensity lipoprotein ; lv , left ventricle ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. relationships between ptx3 and bnp levels and ptx3 levels and lvmi . simple linear regression analysis shows a positive and significant correlation between ( a ) ln(ptx3 ) and ln(bnp ) ( r=0.295 , p<0.001 ) and ( b ) a significant but weak positive correlation between ln(ptx3 ) and ln(lvmi ) ( r=0.114 , p<0.05 ) . bnp indicates btype natriuretic peptide ; lvmi ; left ventricular mass index ; ptx3 , pentraxin 3 . the mean followup period was 30 months ( range : 1 to 59 months ) . during this time , 106 of these patients experienced cardiovascular events , including cardiovascular death ( n=9 ) , nonfatal mi ( n=4 ) , unstable angina pectoris ( n=13 ) , ischemic stroke ( n=9 ) , hospitalization for hf decompensation ( n=41 ) , and coronary revascularization ( n=30 ) . the frequency of cardiovascular events was significantly higher in the highptx3 group ( n=75 ) than in the lowptx3 group ( n=31 , p<0.001 ) ( table 2 ) . hfrelated cardiovascular events , including cardiovascular death and hospitalization for hf decompensation , were significantly higher in the highptx3 group ( n=41 ) than in the lowptx3 group ( n=9 ) ( p<0.001 ) ( table 2 ) . kaplanmeier analysis showed that the probability of cardiovascular events was also significantly higher in the highptx3 group than in the lowptx3 group ( p<0.001 , logrank test ) ( figure 3a ) . cardiovascular events in hfnef patients with low and high plasma ptx3 levels differences between the groups were assessed by the logrank test . hf indicates heart failure ; hfrelated cardiovascular events , cardiovascular death and hospitalization for hf decompensation ; hfnef , heart failure with normal left ventricular ejection fraction ; ptx3 , pentraxin 3 . patients with ( a ) high ptx3 levels and low ptx3 levels , ( b ) subgroups of patients with high and low ptx3 levels plus bnp , and ( c ) subgroups of new york heart association class ii patients with high and low ptx3 levels are shown . the combination of ptx3 and bnp levels led to identification of subgroups ( n=116 , highptx3 and bnp group ; n=64 , highptx3 and lowbnp group ; n=64 , lowptx3 and highbnp group ; and n=116 , lowptx3 and bnp group ) with significantly different probabilities of cardiovascular events ( p<0.001 , logrank test ) ( figure 3b ) . in the lowbnp ( p=0.02 ) and highbnp ( p<0.01 ) groups , the probability of cardiovascular events was significantly higher in those with high ptx3 levels than in those with low ptx3 levels . even among patients with mild hf symptoms ( new york heart association class ii ) , the outcomes were poorer in patients with high ptx3 levels than in those with low ptx3 levels ( p<0.001 , logrank test ) ( figure 3c ) . the results of univariate and multivariable cox proportional hazards analyses for cardiovascular events are shown in tables 3 and 4 . multivariable cox proportional hazards analysis identified ptx3 ( hazard ratio [ hr ] : 1.16 ; 95% ci : 1.05 to 1.27 ; p<0.01 ) , highdensity lipoprotein cholesterol ( hr : 0.99 ; 95% ci : 0.97 to 0.99 ; p<0.05 ) , lvmi ( hr : 1.03 ; 95% ci : 1.05 to 1.27 ) , and bnp ( hr : 1.08 ; 95% ci : 1.03 to 1.14 ; p<0.001 ) as predictors of future cardiovascular events after adjustment for significant factors that were identified by univariate analysis ( table 3 ) . using the forced inclusion model , which evaluated levels of inflammatory markers and bnp ( model 1 ) in multivariable cox hazards analysis , ptx3 , but not hscrp , significantly predicted cardiovascular events ( hr : 1.17 ; 95% ci : 1.07 to 1.28 ; p<0.001 ) . in the forced inclusion model , which included pf5 and bnp , ptx3 significantly predicted cardiovascular events ( model 2 : hr : 1.16 ; 95% ci : 1.05 to 1.27 ; p<0.01 ; model 4 : hr : 1.16 ; 95% ci : 1.06 to 1.27 ; p<0.01 ) ( table 4 ) . furthermore , multivariable cox proportional hazards analysis identified ptx3 ( hr : 1.16 ; 95% ci : 1.01 to 1.35 ; p<0.05 ) and bnp ( hr : 1.21 ; 95% ci : 1.11 to 1.31 ; p<0.001 ) as predictors of future hfrelated cardiovascular events , including cardiovascular death and hospitalization for hf decompensation , after adjustment for significant factors identified by univariate analysis . we classified patients with hfnef into 4 groups according to the levels of ln(ptx3 ) : first quartile ( < 0.59 , ptx3 : < 1.80 ng / ml ) , second quartile ( 0.59 to 1.10 , ptx3 : 1.80 to 3.00 ng / ml ) , third quartile ( 1.10 to 1.35 , ptx3 : 3.00 to 3.85 ng / ml ) , and fourth quartile ( > 1.35 , ptx3 : > 3.85 ng / ml ) ; the relative risk of total cardiovascular events was 1 ( reference ) , 1.36 , 2.71 ( p<0.001 versus the reference ) , and 3.17 ( p<0.001 versus the reference ) , respectively . the logtransformation values of ptx3 demonstrated a linear association with the occurrence of cardiovascular events in patients with hfnef ( r=0.965 , p=0.03 ) . cox proportional hazards analysis of factors that were predictive of future cardiovascular events in patients with hfnef after adjustment for significant factors identified by univariate analysis bnp indicates btype natriuretic peptide ; cad , coronary artery disease ; e / e , mitral early diastolic peak flow velocity to tissue doppler early mitral annular diastolic velocity ; gfr , glomerular filtration rate ; hdl , highdensity lipoprotein ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hr , hazard ratio ; hscrp , highsensitivity creactive protein ; il6 , interleukin6 ; ldl , lowdensity lipoprotein ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. cox proportional hazards analysis of factors that were associated with future cardiovascular events in hfnef patients using forced inclusion models bnp indicates btype natriuretic peptide ; ci , confidence interval ; hf , heart failure ; hfnef , heart failure with normal ejection fraction ; hr , hazard ratio ; hscrp , highsensitive creactive protein ; il6 , interleukin6 ; lvef , left ventricular ejection fraction ; nyha , new york heart association ; ptx3 , pentraxin 3 ; tnf , tumor necrosis factor. we also estimated the cstatistics of pf5 alone . separate incorporation of ptx3 and bnp into pf5 increased the cstatistics for the prediction of future cardiovascular events from 0.617 for pf5 alone to 0.656 for pf5 plus ptx3 and pf5 plus bnp ( table 5 ) . moreover , incorporation of ptx3 and bnp into pf5 increased the cstatistics to 0.683 ( p<0.05 ) . in addition , the pf5 plus bnp plus ptx3 model performed better than the pf5 plus bnp model ( cstatistics : 0.683 versus 0.656 ) . we also confirmed good calibration for the analysis by grnnesby and borgan statistics ( p=0.19 ) . cstatistics for cox proportional hazards analysis predicting future cardiovascular events in patients with heart failure with normal ejection fraction bnp indicates btype natriuretic peptide ; pf5 , the 5 factors identified in the ipreserve study as prognostic in patients with heart failure with normal ejection fraction ( age , presence of diabetes mellitus , previous hospitalization for heart failure , new york heart association classification , and left ventricular ejection fraction ) ; ptx3 , pentraxin 3 . the nri was significant after ptx3 was included : 12.2% for patients without cardiovascular events , 0% for those with cardiovascular events , and 12.2% overall ( p<0.01 ) ( table 6 ) . reclassification of the risk of pf5 and bnp for cardiovascular events after the addition of ptx3 bnp indicates btype natriuretic peptide ; pf5 , the 5 factors identified in the ipreserve study as prognostic in patients with heart failure with normal ejection fraction ( age , presence of diabetes mellitus , previous hospitalization for heart failure , new york heart association classification , and left ventricular ejection fraction ) ; ptx3 , pentraxin 3 . to the best of our knowledge , this report is the first to show a significant association between plasma levels of ptx3 , a marker of inflammation , and adverse cardiovascular outcomes in patients with hfnef . the addition of high ptx3 levels to the pf5 and bnp levels , which were previously found to be prognostic in patients with hfnef , improved their risk stratification , as indicated by a substantial increase in the cstatistics and significance of nri . activation of the inflammatory process is important in the pathogenesis of hf and in adverse prognosis of patients with this condition . several studies have investigated the role of inflammation as a therapeutic target , although initial trials had limited success . consequently , specific antiinflammatory approaches for the different types and stages of hf ( ie , hfnef and hfref ) remain a priority , as does a better understanding of the mechanisms underlying hfrelated inflammation . the established inflammatory marker hscrp is an independent predictor of morbidity and mortality in patients with hf . elevated crp levels predict hospitalization of hf patients , although the association between crp and hf events is no longer significant after adjustment for the presence of lv diastolic dysfunction . consequently , the relationships between inflammatory markers and the prognosis of patients with hfnef remain unclear . , remodeling is driven by the loss of cardiomyocytes , whereas in hfnef , a systemic inflammatory state induces oxidative stress in the coronary microvascular endothelium , which drives myocardial dysfunction and remodeling . other studies have shown that serum levels of inflammatory cytokines are high in patients with hfnef and that interleukin16 , a cytokine considered an important mediator in inflammation , promotes cardiac fibrosis and myocardial stiffening in hfnef . consequently , investigating the relationships between inflammatory markers and prognosis in hfnef is important . we observed a significant correlation between plasma levels of ptx3 , a novel inflammatory maker , and future cardiovascular events in patients with hfnef . furthermore , considerable evidence suggests that ptx3 may better reflect local inflammatory status in tissues than does liverderived crp . we previously reported that ptx3 was partly produced in the coronary circulation in patients with hfnef . these results suggest that ptx3 , rather than crp , reflects vascular and cardiac inflammation in patients with hfnef and might be a cardiovascular biomarker for the assessment and management of hf . in the present study , we showed that increased inflammation as assessed by ptx3 measurement was significantly associated with poor prognosis for hfnef patients by multivariable cox hazards analysis . ptx3 as an inflammation maker and bnp as a myocardial stress marker  to the previously established prognostic factors in the ipreserve study of hfnef patients improved their risk stratification performance , as indicated by an increase in the cstatistics . these results suggest the possible relative merits of these 2 biomarkers for future risk management in hfnef . in nri analyses , the addition of ptx3 was able to provide a more appropriate risk assessment in patients who were evaluated as having a high risk state by previously described hfnef prognostic factors ( pf5 plus bnp ) in patients without cardiovascular events . furthermore , the addition of ptx3 never resulted in a worse risk classification in patients with cardiovascular events . consequently , the significance of nri was shown by incorporating ptx3 into the new riskassessment model . the site of ptx3 production in patients with hfnef remains unclear . under normal physiological conditions , ptx3 is expressed in atherosclerotic lesions , adipose tissue , and the heart with acute and subacutephase hf ; however , the types of cells that express ptx3 in failing hearts of patients with hfnef and chronic hf have not been determined . accumulation of the extracellular matrix ( ie , fibrosis ) may also be pathophysiologically important in progression of the hfnef disease process and be a contributor to subsequent events . hfnef may also be caused by structural and molecular abnormalities of the cardiovascular system , including cardiac and noncardiac factors , such as those associated with vascular functions . we recently demonstrated significant involvement of endothelial dysfunction in the prognosis of patients with hfnef , suggesting that improvement in endothelial function could be a potential therapeutic target in patients with hfnef . ptx3 is produced by fibrous tissues and endothelial cells in response to inflammatory stimuli and is considered an important marker of vascular pathology . in patients with acute mi and infectious myocarditis , ptx3 is produced by macrophages , endothelial cells , and , to a lesser extent , myocardiocytes and is localized in the interstitium . the interstitial localization of ptx3 in human failing hearts suggests that ptx3 is produced locally by fibroblasts in the cardiac interstitium . consequently , in patients with hfnef , ptx3 might be produced by myocardial fibroblasts and/or endothelial cells rather than in subclinical atherosclerotic lesions in patients with hfnef . we found that the frequency of hfrelated cardiovascular events , including cardiovascular death and hospitalization for hf decompensation , was significantly higher in patients with high ptx3 levels than in those with low ptx3 levels . this finding suggests that ptx3 is more predictive of future hfrelated cardiovascular events than of vascular events in patients with hfnef . we used the median value of ptx3 ( 3.0 ng / ml ) as the cutoff point in the present study . suzuki et al demonstrated that the median value of ptx3 was 3.7 ng / ml in patients with hfref and split the patients into 2 groups : those above and those below 4.0 ng / ml . in this study , patients with hfref were targeted , and it has already been demonstrated that patients with hfref have higher inflammatory activity than those with hfnef . we previously demonstrated that the 75% range value of ptx3 was 2.90 ng / ml in nonhf patients with risk factors ( hypertension , 67.8% ; diabetes mellitus , 37.4% ; dyslipidemia , 57.1% ; and coronary artery disease , 55.0% ) . furthermore , inoue et al showed that ptx3 levels were < 2.28 ng / ml in healthy volunteers . in previous studies of cardiovascular events in stable patients with coronary risk factors , thus , we consider that the median value of 3.0 ng / ml for ptx3 was a clinically meaningful cutoff point in our study . first , our study included only a relatively small number of patients in a single center . second , the population of this study was relatively young and predominantly male ; had a low prevalence of atrial fibrillation , a higher frequency of mild hf , relatively lower bnp levels ; and had less frequent use of diuretics . because our institution is an educational hospital and the design was a singlecenter study in japan , the recruited patients with hfnef might have had differences in characteristics compared with those in western studies . consequently , a large , multiracial , multicenter study is required to confirm our results . recent studies in mouse models have shown that ptx3 has a cardioprotective effect , suggesting that ptx3 might protect the cardiovascular system by modulating the immune  inflammatory balance . further in vivo and in vitro experiments are required to determine the exact role of high ptx3 levels in hfnef . despite these limitations , a largescale multicenter trial is warranted to further examine the pathological role and clinical significance of ptx3 in hfnef . high plasma ptx3 levels , but not other inflammatory markers including hscrp , are significantly correlated with future cardiovascular events in patients with hfnef . we are grateful to megumi nagahiro and saeko tokunaga of the department of cardiovascular medicine , faculty of life sciences , kumamoto university , kumamoto , japan , for their skillful technical assistance .
backgroundpentraxin 3 ( ptx3 ) is a novel inflammatory marker produced by various cell types including those of the vasculature and the heart . 
 the relationship between inflammatory markers and prognosis of patients with heart failure with normal ejection fraction ( hfnef ) remains unknown . 
 we investigated whether plasma ptx3 levels can predict future cardiovascular events in patients with hfnef.methods and resultsplasma ptx3 , highsensitivity creactive protein , and btype natriuretic peptide levels were measured prospectively in 360 stable patients with hfnef . 
 the subsequent incidence of cardiovascular events , including cardiovascular death , nonfatal myocardial infarction ( mi ) , unstable angina pectoris , nonfatal ischemic stroke , hospitalization for heart failure decompensation , and coronary revascularization , was determined . during a mean 30month followup , 
 106 patients experienced cardiovascular events . 
 these events were more frequent in patients with high plasma ptx3 levels ( > 3.0 ng / ml ) than low levels ( 3.0 ng / ml ) . 
 multivariable cox hazard analysis showed that ptx3 ( hazard ratio : 1.16 ; 95% ci : 1.05 to 1.27 ; p<0.01 ) and btype natriuretic peptide ( hazard ratio : 1.08 ; 95% ci : 1.03 to 1.14 ; p<0.001 ) , but not highsensitivity creactive protein levels , were significant predictors of future cardiovascular events . 
 multivariable cox analysis with the forced inclusion model , including 5 previously identified prognostic factors , found that ptx3 was a significant predictor of cardiovascular events ( hazard ratio : 1.16 ; 95% ci : 1.06 to 1.27 ; p<0.01 ) . 
 the cstatistics for cardiovascular events substantially increased from 0.617 to 0.683 when ptx3 was added to the 5 previously identified prognostic factors.conclusionshigh plasma ptx3 levels , but not other inflammatory markers , are correlated with future cardiovascular events in patients with hfnef . 
 ptx3 may be a useful biomarker for assessment of risk stratification in hfnef.clinical trial registrationurl : http://www.umin.ac.jp ; unique identifier : umin000002170 .