
 pulmonary hypertension ( ph ) encompasses a diverse group of disorders , all characterized by an elevation in pulmonary arterial pressure and pulmonary vascular resistance ( pvr ) .
pediatric ph is associated with significant morbidity and mortality , and differs from ph as manifest in adults in several important ways , such as the development of ph in a growing lung .
medical treatment of pediatric ph has been challenging due to a lack of accessible , noninvasive , objective measures that can aid in managing these patients .
specifically , there is a need for better tools for prognosis and assessment of the progression of the disease [ 1 , 3 , 4 ] .  
outcomes of most interest to clinicians and ph patients include death , hospitalization for ph - related events , right ventricular failure , initiation of intravenous prostanoid therapy , and lung transplantation .
although definitive , these end points are often difficult to study in pediatric ph due to their infrequent occurrences and the requirement for long - term followup . currently , disease progression is most commonly assessed through a 6-minute walk distance and hemodynamic assessments , but both of these suffer from practical drawbacks and neither is thoroughly validated in the pediatric population .
these limitations in current outcomes and prognostic variables have spurred our search for reliable , noninvasive , objective , and cost - effective alternatives that are easily measured in children [ 1 , 4 ] .
circulating protein biomarkers have the potential to meet these objectives . the role of inflammation in the pathobiology of ph has recently been emphasized [ 69 ] .
cytokine- and growth factor - dependent mechanisms lead to inflammatory cell recruitment and are prominent in ph [ 911 ] . of these factors ,
for example , il-6 administration in rats induces ph , and il-6 augments hypoxia - induced ph .
in addition , it has been reported recently that transgenic mice overexpressing il-6 display ph , vascular remodeling , and an exaggerated response to hypoxia .
further , circulating levels of cytokines are reportedly important in the pathogenesis of a wide range of other conditions including chronic heart failure , acute renal failure , and sepsis .
recent developments in protein array technology allow high throughput , multiplex analysis with excellent sensitivity , precision , and specificity .
the approach is also cost effective and uses minimal sample because all the analytes are measured on a single chip simultaneously .
the requirement for low sample volumes is of special interest in the management of pediatric patients .
we therefore applied a protein array approach that provides accurate and precise quantification of twelve proteins with known lung or vascular effects in a cohort of pediatric patients with ph .
our objective was to determine whether there is a clinically relevant association between any of the panel constituents , individually or combined , with hemodynamic parameters , disease prognosis , and relevant adverse outcomes .
prior approval for these studies was obtained from the colorado multiple institutional review board ( comirb , approval # 05 - 0551 ) . written informed consent
was obtained from the parents or guardians of all children who served as subjects of the investigation , and when appropriate , from the subjects themselves .
plasma samples were collected from 70 pediatric patients with ph ranging in age from newborn to 21.3 years old .
all patients were being treated with appropriate therapies to manage their ph during sample collection ( table 1 ) .
patients were classified as idiopathic pediatric pulmonary arterial hypertension ( ipah ) or associated pulmonary arterial hypertension ( apah ) according to published criteria .   in this study , disease prognosis / severity was determined using hemodynamic parameters measured in the cardiac catheterization laboratory during a routine pulmonary hypertension visit .
the data were obtained with the patient breathing room air , prior to testing pulmonary reactivity with the use of vasodilator therapy . for right - heart catheterization
, we used a swan - ganz catheter in the femoral vein or internal jugular vein to determine mean right atrial pressure ( rap ) , mean pulmonary artery pressure ( pap ) , pulmonary capillary wedge pressure ( pcwp ) , cardiac index ( ci ) , and pulmonary vascular resistance index ( pvri ) .
invasive arterial monitoring was used to measure the systemic vascular resistance index ( svri ) .
we used fick with assumed oxygen consumption in those patients with shunts and thermodilution in all others to measure cardiac output and then calculate cardiac index .
most subjects were ventilated because they were children . in addition , the reactivity to inhaled nitric oxide was calculated by taking the difference in mean pap between room air and with nitric oxide . the adverse outcome used in the predictive models
was defined as initiation of intravenous prostanoids ( epoprostenol or treprostinil ) , transplantation , and/or death occurring within 12 months from the collection of the blood sample .
admission for right heart failure is a rare event in children and was not included in the adverse outcome definition .  
blood ( 5  ml ) was drawn , plasma isolated , and this was frozen at 70c until analysis .
protein determinations were performed on an evidence analyzer ( randox laboratories , northern ireland ) according to the manufacturer 's protocol for the simultaneous quantification of the following analytes : interleukin-1alpha ( il-1 ) , interleukin-1beta ( il-1 ) , interleukin-2 ( il-2 ) , interleukin-4 ( il-4 ) , interleukin-6 ( il-6 ) , interleukin-8 ( il-8 ) , interleukin-10 ( il-10 ) , tumor necrosis factor alpha ( tnf ) , gamma interferon cytokine ( ifn ) , monocyte chemotactic protein-1 ( mcp-1 ) , endothelial growth factor ( egf ) , and vascular endothelial growth factor ( vegf ) .
values for individual proteins measured by this multiplexed protein array technology have been shown to correlate with single elisa measurements .
further , intra- and interassay coefficients of variation of less than 10% are typical for most protein measured by this multiplexed approach with this system .
determinations were made in duplicate and all calculations were based on the average of the two measurements for each sample .
descriptive statistics were calculated using mean  standard deviations or medians and the interquartile range ( iqr ) for continuous variables and percentages for categorical variables .
comparisons across groups were made by wilcoxon rank sum or chi - squared tests , as appropriate .
spearman rank correlation coefficients were used to estimate the association between the hemodynamic variables and protein markers .
logistic regressions were fit to investigate the association between the dichotomous adverse outcome variable ( outcome observed within 12 months ) and the protein measurements .
mean standardization and principal component analysis ( pca ) were performed for dimension reduction across the protein measurements , resulting in a single value representing a weighted combination of all twelve markers .
a score statistic was used to select the principal component ( pc ) most associated with the outcome and the best subset of two pcs and clinical variables in a multivariate logistic regression .
the predictive ability of the model was investigated using a c - index ( area under the roc curve ) .
the improvement in risk prediction associated with the addition of the protein biomarker pc was assessed by calculation of a net reclassification improvement ( nri ) measure .
to estimate the predictive ability of the logistic regressions to an independent set of observations , c - indices were calculated using the leave - out - one cross - validation approach .
all analyses were performed using sas version 9.2 software ( sas institute inc . , cary , nc , usa , 2008 ) .
the study population consisted of 70 pediatric patients with ph : 36% ipah and 64% with apah . of these ,
the median pap was elevated at 34  mm  hg ( the interquartile range ( iqr ) was 2356  mm  hg ) with a median rap value of 5  mm  hg ( iqr , 37  mm  hg ) .
nine ( 13% ) patients experienced an adverse event within 12 months of sample collection .
these patients had worse mpap levels ranging from 30 to 76 with a median of 65.5  mm  hg as well as a median pvri of 15.5 wood units  m ranging from 5.120.4 .
only 39 of the 48 patients with vasoreactivity tests had complete data in order to assess the responder status based on the pediatric definition .
based on these data 2 ( 5% ) were classified as responders and neither of these patients experienced an adverse event .
there were no significant differences between the ipah and apah groups for any of the protein levels .  
thereafter , only 59 ( 84% ) of the patients had a catheterization performed on the same day as blood collection for the correlation analysis , and of these , 48 individuals also had a vasoreactivity test in response to inhaled nitric oxide ( no ) .
the following pairs of hemodynamic and circulating protein markers had correlation coefficients ( r ) significantly different from zero : il-1 and ci ( r = 0.31 , cl = 0.53 to 0.04 ) ; egf and mpap ( r = 0.45 , cl = 0.63 to 0.21 ) ; egf and pvri ( r = 0.37 , cl = 0.57 to 0.12 ) ; egf and pvr / svr ( r = 0.42 , cl = 0.62 to 0.17 ) ; il-6 and mpap ( r = 0.27 , cl = 0.01 to 0.49 ) ; il-6 and pvri ( r = 0.32 , cl = 0.06 to 0.53 ) .
none of the biomarkers correlated with the change in mean pap in response to inhaled no .   to assess the potential additive predictive ability of the proteins we measured ,
the first of these were univariate models that were estimated to determine the association of each protein individually with the adverse outcome variable .
vegf and il-6 were significantly associated with the adverse outcome : ( or ( 95% ci ) were as follows : 0.56 ( 0.330.98 ) and 1.69 ( 1.032.77 ) , respectively .
in addition to vegf , il-1 , il-2 , il-4 , mcp1 , and egf also had negative associations , indicating that lower values of these proteins were associated with the adverse event outcome .
pca was performed on the protein marker data to condense the 12 measurements to a few orthogonal components .
the 3rd component was significantly associated with the outcome , with higher values corresponding to an increased risk of an adverse event .
the risk associated with this protein index was weighted by higher values of il-1 and il-6 and lower vegf values ( figure 2 ) .
this finding corresponds well with the results from the univariate analyses and gave a c - index of 0.81 .
score statistics were used to identify the top predictive clinical markers and determine the added value of a protein marker index over the top clinical predictor . the resulting clinical model identified pap as the top predictor . using this model as a base model , the pcs of the protein measurements were added as predictors and the selection process was repeated .
after mpap , the 4th pc was the top predictor indicating it contains information orthogonal to the hemodynamic variable .
the risk associated with the 4th pc was weighted by higher values of mcp1 and il-6 and lower il-10 values . to evaluate the added predictive value of the protein measurements in addition to mpap , roc curves , corresponding c - indices , and nri measures were utilized ( figure 3 ) .
the inclusion of these two proteins increased the c - index from 0.81 to 0.90 .
significant improvement was seen with the addition of the protein measurements ( nri p value = 0.01 ) indicating that their inclusion enhanced the models ability to correctly classify patients .
this result was verified with a resampling approach used to estimate the models predictive ability when applied to an independent set of observations .
the estimated c - indices for the model with mpap alone , and with mpap and the protein measurements , were 0.69 and 0.82 , respectively .
our data ( table 1 ) indicate a surprisingly high morbidity and mortality , even in children with mild pulmonary hypertension , and they underscore the fact that this is a very high - risk group .
this observation stimulated our efforts to develop a practical and noninvasive approach to the management of pediatric pulmonary hypertension that can be used in a routine setting .
of interest and clinical relevance is the fact that we have shown that by simply adding the quantification of a set of plasma proteins , it is possible to markedly improve our ability to predict outcomes in children with ph .
specifically , the addition of a protein index , weighted mostly by il-6 , il-10 , and mcp-1 concentrations , significantly increased the probabilities for those patients with an adverse event compared to hemodynamic measurements alone .
when investigating the plasma proteins univariately , vegf and il-6 were associated with occurrence of an adverse event and egf and il-6 were correlated with mean pap and pvri .
in addition , we identified a combination of all twelve proteins which associated well with adverse events and had similar predictive ability compared to the top hemodynamic predictor .  
interest in the role of cytokines and growth factors in the development of ph has grown .
recent evidence is consistent with the view that circulating factors and inflammatory cells contribute to remodeling in chronic ph [ 11 , 24 , 25 ] .
circulating and lung levels of the proinflammatory cytokine il-6 are increased in patients with idiopathic ph and ph associated with inflammatory diseases [ 1215 ] .
il-6 may also be a mediator of disease in ph because overexpression of il-6 in a mouse model exacerbated ph by both proproliferative and antiapoptotic mechanisms .
for example , il-6 treated mice are prone to develop hypoxia - induced pulmonary hypertension   which may be mediated in part by an inflammatory process . in our study
, we found that increased levels of circulating il-6 were significantly associated with an increased risk of an adverse event but when considered multivariately with pap , ci , and vegf , it was replaced with an anti - inflammatory cytokine , il-10 .
recently there has also been increased interest in the role of vegf in pulmonary arterial hypertension .
vegf is responsible for angiogenesis and vasculogenesis , and animal models indicate that when vegf signaling is impaired , this contributes to the pathogenesis of ph [ 28 , 29 ] .
alternatively , growth factors may be important for the maintenance of continued lung growth in patients with pulmonary arterial hypertension , and a favorable phenotype may be associated with increased vegf and egf .
farkas et al .   found a similar inverse relationship between vegf and pap in rats and lassus et al .  
the search for biomarkers that are useful in the management of pediatric ph is in its infancy , but the ideal marker would be obtained noninvasively , easily measured , and would offer high sensitivity and specificity . currently , the brain natriuretic peptide system and neurohumoral markers have been evaluated in children with ph [ 3234 ] .  
our study demonstrates that quantification of a panel of cytokines and growth factors has potential applications in clinical trial design by identifying patients at risk for an adverse event .
in addition , patients at higher risk for an adverse event may need to be treated more aggressively with continuous intravenous therapy or listed for lung transplantation .
this study adds to the growing body of literature indicating that inflammation is important in pediatric ph and that circulating ( blood ) biomarkers can be important tools in prediction and prognostic evaluation of pediatric patients with ph .
the potential for growth factors , egf and vegf , to aid in the management of ph is important and novel .
serial sampling and assessment of longitudinal changes might provide important additional insights , but these findings indicate that even a single point in time determination can predict the future clinical course .
we intentionally examined a heterogeneous group of patients in this study in order to explore patterns of biomarker expression .
it is clear that the next step is to validate these findings in a larger scale study including several groups of well - characterized subjects .
we suggest that , based on our findings , additional evaluation of cytokines and growth factors is warranted because there is growing evidence that these determinations may be relevant for disease management .
 
 background . management of pediatric pulmonary hypertension ( ph ) remains challenging . 
 we have assessed a panel of circulating proteins in children with ph to investigate their value as predictive and/or prognostic biomarkers . from these determinations 
 , we aim to develop a practical , noninvasive tool to aid in the management of pediatric ph . methods . 
 twelve cytokines and growth factors putatively associated with lung or vascular disease were examined in plasma specimens from 70 children with ph using multiplex protein array technology . 
 associations between hemodynamics , adverse events , and protein markers were evaluated . 
 results . 
 epidermal growth factor ( egf ) and il-6 were associated with important hemodynamics . of the twelve proteins , vegf and il-6 
 were significantly , univariately associated with the occurrence of an adverse event , with odds ratios ( 95% confidence intervals ) of 0.56 ( 0.330.98 ) and 1.69 ( 1.032.77 ) , respectively . when hemodynamic predictors were combined with protein markers , the ability to predict adverse outcomes within the following year significantly increased 
 . conclusions . 
 specific circulating proteins are associated with hemodynamic variables in pediatric ph . 
 if confirmed in additional cohorts , measurement of these proteins could aid patient care and design of clinical trials by identifying patients at risk for adverse events . 
 these findings also further support a role for inflammation in pediatric ph . 