The pro-apoptotic serum activity is an heart failure

European Heart Journal (2004) 25, 1620–1625
Clinical research
The pro-apoptotic serum activity is an
independent mortality predictor of patients with
heart failure
¨ssiga, Stephan Fichtlscherera, Christopher Heeschena
Lothar Ro
¨rgen Bergerb, Stefanie Dimmelera,*, Andreas M. Zeihera
Ju
a
Division of Molecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai
7, 60590 Frankfurt, Germany
b
Institute of Mathematics and Computer Science in Medicine, University of Hamburg, Hamburg, Germany
Received 19 January 2004; revised 9 June 2004; accepted 24 June 2004
Available online 20 August 2004
Aim Systemic inflammation with elevated serum levels of circulating pro-inflammatory cytokines is a major determinant of prognosis in heart failure (HF). Since serum
of patients with HF induces apoptosis of endothelial cells (EC), we aimed to determine whether the pro-apoptotic activity in the serum may predict prognosis of patients with HF.
Methods and results We measured the pro-apoptotic activity in the serum of 48 patients with HF of different aetiology by an ex vivo cell culture assay and subsequently
monitored these patients for the single endpoint all-cause mortality. During followup, 16 patients died and 11 patients received a heart transplant. Survivors had a lower
pro-apoptotic serum activity (P = 0.007). By univariate analysis, pro-apoptotic serum
activity, NYHA class, pro-BNP, low blood pressure, and creatinine levels were significantly associated with mortality. In a multivariable stepwise Cox-regression model,
the pro-apoptotic serum activity (adjusted hazard ratio, HR = 1.85 per %, P = 0.008),
elevated pro-BNP levels (HR = 9.35 per log[pro-BNP], P = 0.001), and low blood pressure
(HR = 0.96 per mmHg, P = 0.041) remained as independent predictors of death.
Conclusion In this exploratory study, the pro-apoptotic serum capacity is independently
associated with a worse prognosis in patients with HF, suggesting that the assessment
of serum-induced EC apoptosis could provide an integrative estimate of the deleterious
effects of various pro-inflammatory cytokines and other cytotoxic factors in HF.
c 2004 The European Society of Cardiology. Published by Elsevier Ltd. All rights
reserved.
Introduction
Current understanding of the pathophysiology of advanced heart failure (HF) implicates neurohumoral acti-
* Corresponding author. Tel.: +49-696-301-7440; fax: +49-696-3017113.
E-mail address: [email protected] (S. Dimmeler).
vation as an important prognostic component.1 Levels
of circulating catecholamines and circulating cytokines
including tumour necrosis factor a (TNFa), its soluble
receptors 1 and 2 (sTNF-R1 and -R2), soluble CD14
(sCD14), or interleukin-6 (IL-6) were shown to be associated with an unfavourable outcome in patients with
HF.2,3 Cytokines are well-established to induce apoptotic
cell death of endothelial cells (EC).4 Importantly, previ-
0195-668X/$ - see front matter c 2004 The European Society of Cardiology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.ehj.2004.06.036
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KEYWORDS
Heart failure;
Prognosis;
Apoptosis;
Serum marker;
Endothelial cells
The pro-apoptotic serum activity
1621
ous studies have demonstrated that the serum of patients with HF is pro-apoptotic for EC.5–7 We, therefore,
hypothesised that the pro-apoptotic activity in the serum
of patients with HF may quantitatively integrate the
cytotoxic insult to EC by neurohumoral activation and,
as a functional read-out of the entire cascade of pro-inflammatory cytokines, may affect disease progression in
patients with advanced HF.
Methods
Patients
Table 1 Clinical characteristics and medical treatment of
48 CHF patients at inclusion
Age (years)
Gender (male/female)
NYHA class (II/III/IV)
Aetiology (ischaemic/non-ischaemic)
LVEF (%)
LVEDD (mm)
Intraventricular conduction
delay/QRS duration (ms)
Rhythm (Sinus/A. fib./Pacemaker)
57 ± 1
37/11
20/21/7
22/26
25 ± 1
65 ± 1
23 (48)/128 ± 5
34/12/2
Medical treatment
ACE-Inhibitor/AT1 Receptor Blocker
b-Blocker
Spironolactone
Diuretics
Digitalis
Amiodarone
Statin
Implanted cardioverter/defibrillator
Surgery (LV reduction/assist devices)
48 (100)
20 (42)
11 (23)
43 (90)
36 (75)
18 (38)
11 (23)
11 (23)
1 (2)/2 (4)
Serum parameters
pro-BNP (pg/ml)
TNFa (pg/ml)
sTNF-R1 (pg/ml)
Apoptosis (%)
3666 ± 595
3.6 ± 0.7
1670 ± 112
4.3 ± 0.2
Haemodynamics
Mean arterial blood pressure MAP (mmHg)
85 ± 2
Numbers of patients (% of all patients) or mean ± SEM.
Definition of endpoint
Patients were monitored via our outpatient and heart transplant
programme at standard intervals over a minimum follow-up of
30 months (median follow-up for survivors 1254 days). All-cause
mortality was the single endpoint. Patients, who received a
heart transplant during follow-up, were considered as survivors
for the time period from inclusion to the date of transplantation
with no further follow-up beyond this point of time (‘‘censored
alive’’).
Ex vivo pro-apoptotic activity assay
As previously reported in detail,6 the systemic pro-apoptotic
activity was assessed by an ex vivo cell culture assay. In
brief, human umbilical venous endothelial cells (HUVEC, Cell
Systems/Clonetics, Solingen, Germany) were grown in endothelial basal medium (EBM) supplemented with hydrocortisone,
bovine brain extract, gentamicin, amphotericin B, epidermal
growth factor, and 10% foetal calf serum until the third passage before experiments were performed. For quantification
of the pro-apoptotic activity of patient serum, cell culture
medium was removed, cells were washed, and subsequently
incubated with a modified medium, which contained 10% patient serum. Following 18 h of incubation, nuclei were
stained and the pro-apoptotic activity of the serum was quantified as the percentage of apoptotic nuclei out of the total
number of cells. Two independent, blinded investigators assessed features of nuclear morphology characteristic for
apoptotic cells by fluorescence microscopy as described previously.4 The reproducibility of these measurements was determined in a subset of individuals (n = 20 patients and healthy
subjects) by the comparative analysis of two aliquots of the
same serum sample in two individual HUVEC passages from
the same lot. The intra-sample inter-HUVEC correlation of
these measurements was r = 0.86, P < 0.001. For the inter-observer variability between two blinded investigators, the correlation was r = 0.77, P < 0.001 in n = 63 individuals (patients
and healthy subjects).
Cytokine and pro-BNP assays
TNFa and soluble TNF receptor 1 (sTNF R1) serum concentrations were measured by enzyme-linked immunosorbent assays
(ELISA, R & D Systems, Wiesbaden). NT-pro-brain natriuretic
peptide (pro-BNP) was determined by ELISA (Roche Diagnostics).
Statistics
Data are expressed as mean ± SD or as indicated. Cumulative
survival was univariately evaluated by Kaplan–Meier analysis
(log-rank test). Hazard ratios (HR) were calculated by univariate
Cox regression analysis. Factors with a strong univariate significance indicated by a P-value below 0.005 were stepwise included in a multivariable Cox regression model in order to
adjust factors for their interdependency. Individual parameters
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Forty-eight Patients with HF were recruited between June 1998
and August 2000 via our outpatient HF clinic at the occasion of
routine visits or when referred for elective, invasive examinations in order to proceed with an evaluation for heart transplantation. At the time of inclusion, patients were on a titrated
medical regimen (see Table 1). Patients with a history of myocardial infarction (MI) within three months before blood sampling as well as patients with decompensated renal
insufficiency (defined by serum creatinine levels P2 mg/dl) or
concomitant infectious or primary pulmonary disease were excluded. Ischaemic aetiology was defined by the presence of significant coronary artery disease documented by coronary
angiography and a history of previous MI. Venous blood samples
were drawn into 10 ml serum tubes without additives, and, following centrifugation, serum aliquots were frozen at 80 °C. All
patients included in this study gave written, informed consent.
The study protocol was approved by the Ethics Committee of
the Johann Wolfgang Goethe University of Frankfurt.
1622
Results
100
80
Survival (%)
that failed to reach a significance level of P < 0.05 when comparatively analysed in this model were omitted from further multivariable analysis. The assumption of proportional hazards was
validated by defining a time-dependent covariate as a function
of time (T) and individual covariates. The assumption of linearity was tested by fractional polynomials. No correction has been
made for multiple hypothesis testing since this, because of the
small sample and endpoint size, was an exploratory study
searching for parameters requiring confirmation in subsequent
adequately powered studies. Statistical significance was
assumed, if the null hypothesis could be rejected at
P = 0.05. All analyses were performed with SPSS software
(version 11.5).
L. Ro
¨ssig et al.
P < 0.02
log-rank test
60
40
20
0
400
800
1200
1600
Days
Clinical and serological characteristics of
patients at baseline
Baseline clinical characteristics, the pro-apoptotic serum
activity as well as cytokine and pro-BNP levels of the 48
patients are summarised in Table 1. The pro-apoptotic
serum activity, as well as serum pro-BNP and cytokine
levels, were correlated with disease severity (data not
shown).
Sixteen patients died during follow-up at a median time
period of 289 days (interquartile range 676) after inclusion. Eleven patients received a heart transplant after
a median of 217 days (interquartile range 262) following
inclusion, at which time these patients were censored as
survivors.
In the subgroup of patients without heart transplants,
baseline serological and clinical parameters were as follows: the pro-apoptotic serum activity at inclusion was
4.97 ± 1.11% in patients with a lethal outcome vs.
3.97 ± 0.98% in those surviving without heart transplantation. Patients, who subsequently died, had pro-BNP levels of 5825 ± 4259 pg/ml and sTNF-R1 levels of
2105 ± 875 pg/ml, whereas in patients, who survived
without heart transplantation, pro-BNP was 1712 ± 3465
pg/ml and sTNF-R1 was 1238 ± 455 pg/ml. Serum levels
of TNFa were 3.6 ± 1.2 pg/ml in patients, who died,
and 4.1 ± 6.9 pg/ml in patients surviving without heart
transplantation.
According to clinical symptoms, patients, who died
during follow-up, had serum creatinine levels of
1.4 ± 0.4 mg/ml and a serum sodium concentration of
138 ± 4 mM vs. 1.1 ± 0.2 mg/ml and 140 ± 3 mM, respectively, in patients surviving without heart transplantation. The echocardiographic left ventricular ejection
fraction (LVEF) was 22 ± 8% in those, who subsequently
died, compared to 28 ± 7% in patients, who survived
without receiving a heart transplant. In this subgroup,
patients who died, had a baseline mean arterial blood
pressure (MAP) of 78 ± 12 mmHg, whereas the MAP was
93 ± 10 mmHg at inclusion in those, who survived without
heart transplantation.
Predictors of mortality
Dichotomisation of the patient population according to
the pro-apoptotic serum activity revealed a significantly
higher mortality of patients with pro-apoptotic activity
above the median value as assessed by Kaplan–Meier
analysis (Fig. 1).
In order to test the predictive value of various risk
factors in our study population for the occurrence of
death within 30 months, we analysed the data by univariate Cox regression. The pro-apoptotic serum activity
significantly influenced survival in HF patients, in addition to established prognostic factors such as the severity
of clinical symptoms according to NYHA classes, levels of
pro-BNP, blood pressure, and serum creatinine concentrations (Table 2). Age, echocardiographic LVEF, tachycardia (defined as a heart rate above 90 bpm),
hyponatraemia (defined as a serum sodium concentration
below 135 mM), and hyperuricaemia (serum uric acid
above 9.5 mg/ml8) were weakly associated with a worse
outcome, but did not reach statistical significance to
predict mortality in the tested cohort.
Independent predictors of mortality
To determine independent predictors of mortality among
these variables, we used a stepwise multivariable model
of regression analysis including every factor with a univariately significant influence on mortality. Among
these, high pro-apoptotic serum activity, elevated BNP
levels, and low blood pressure remained the only
independent predictors of death in patients with HF
(Table 3).
By ROC curve analysis, both the sensitivity and specificity of the pro-apoptotic serum activity to predict mortality (AUC 0.756) were comparable to those for pro-BNP
serum levels (AUC 0.787, Fig. 2).
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Clinical outcome
Fig. 1 Kaplan–Meier cumulative survival analysis of 48 patients with HF
according to the baseline pro-apoptotic serum activity. Continuous line,
patients with pro-apoptotic serum activity below median; broken line,
patients with pro-apoptotic activity serum above median.
The pro-apoptotic serum activity
1623
Table 2 Univariate Cox regression analysis of the predictive value of prognostic factors for all-cause mortality in 48 patients with
HF
Variable
Hazard ratio (95% CI)
P-value
Apoptosis per %
NYHA per class
Age per year
pro-BNP*
LVEF per %
Tachycardic heart rate ( P 90 bpm)
Blood pressure per mmHg MAP
Hyponatraemia (S-Na+ 6 135 mM)
Hyperuricaemia (UA P 9.5 mg/dl)
Serum Creatinine per mg/dl
b-blocker intake
1.97
3.26
1.05
7.76
0.94
2.52
0.94
2.26
2.59
5.28
0.23
0.002
0.004
0.050
<0.001
0.060
0.067
0.002
0.175
0.066
0.002
0.022
(1.30–2.99)
(1.46–7.27)
(1.00–1.16)
(2.63–22.86)
(0.89–1.00)
(0.94–6.75)
(0.90–0.98)
(0.70–7.31)
(0.94–7.13)
(1.84–15.14)
(0.07–0.81)
Parameters were analysed as continuous variables or as indicated. *, logarithmic transformation.
Table 3 Stepwise multivariable Cox regression analysis of the predictive value of prognostic factors for all-cause mortality in 48
patients with HF
Step I
Variable
Hazard ratio (95% CI)
P-value
0.278
0.005
Step II
P-value
0.002
0.112
Variable
Hazard ratio (95% CI)
pro-BNP per log (pro-BNP)
6.61 (2.05–21.29)
Serum Creatinine per mg/dl
2.23 (0.83–6.02)
When log (Pro-BNP) and serum creatinine were considered together, serum creatinine was omitted (P = 0.101)
Step III
Variable
Hazard ratio (95% CI)
P-value
pro-BNP per log (pro-BNP)
9.18 (2.52–33.41)
0.001
Blood pressure per mmHg MAP
0.95 (0.91–0.99)
0.011
When log (Pro-BNP) and blood pressure were considered together, both parameters remained independent significant predictors
of mortality
Step IV
Variable
Hazard ratio (95% CI)
pro-BNP per log (pro-BNP)
9.35 (2.42–36.10)
Blood pressure per mmHg MAP
0.96 (0.92–1.00)
Apoptosis per %
1.83 (1.17–2.86)
If apoptosis was forced into the model, it further improved the fit of the model indicating the independent predictive
this variable for all-cause mortality
P-value
0.001
0.041
0.008
value of
Parameters with a significant predictive value for mortality in univariate analysis were stepwise included in a multivariable Cox regression model.
Discussion
Our data demonstrates for the first time that the proapoptotic serum activity predicts mortality in patients
with HF. Importantly, the pro-apoptotic serum activity
remained a significant predictor of mortality following
stepwise multivariable regression including various clinical, serological, and routine laboratory characteristics of
patients with HF. In this model, the predictive capacity
of the pro-apoptotic serum activity was comparable to
the strong serological prognosticator of mortality in HF
(pro-BNP serum levels) as well as to the leading clinical
symptom of the low output syndrome, hypotensive blood
pressure.
The prognostic relevance of the pro-apoptotic serum
activity as a quantitative marker of endothelial activation indicates the potential pathophysiological contribution of systemic vascular inflammatory processes to the
progression of HF beyond haemodynamic alterations
and independent of the aetiology of cardiac dysfunction.
While the extent and composition of the pro-inflammatory, oxidative and general cytotoxic burden in HF may
relate to both cardiac and peripheral vascular dysfunction, the ex vivo assay used in our study clearly displays
the capacity of these factors to induce apoptosis in EC.
The independent prognostic impact of this pro-apoptotic
activity towards EC underscores an important pathophysiological link, through which the pro-apoptotic serum
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NYHA per class
1.66 (0.67–4.11)
pro-BNP per log (pro-BNP)
5.66 (1.69–18.95)
When both NYHA class and log (Pro-BNP) were included, NYHA was omitted (P = 0.275)
1624
L. Ro
¨ssig et al.
1. 0
Sensitivity
0. 8
0. 5
0. 3
Pro-apoptotic activity
pro-BNP levels
0
0
0. 3
0. 5
0. 8
1 - Specificity
1. 0
Fig. 2 Receiver operator characteristics curve analysis. Parameters
were evaluated for the prediction of mortality in patients with HF.
Continuous line, pro-apoptotic serum activity; broken line, serum proBNP levels.
Acknowledgements
We are grateful to Marga Mu
¨ller-Ardogan for expert
assistance and to Dr. V. Mitrovic (Kerckhoff Heart Center, Bad Nauheim, Germany) for determination of proBNP. The Heinrich und Fritz Riese-Stiftung supported this
work.
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load exerts its deleterious action during HF progression.
However, as in vitro read-out systems to study the induction of cardiomyocyte apoptosis by human serum are not
available, we cannot rule out that sera of patients with
HF may likewise exert a pro-apoptotic activity towards
cardiomyocytes, and that such an effect would also influence prognosis in HF. Further studies are necessary to
analyse the individual pathophysiological role of various
tissue activation by inflammatory stimuli in HF.
Our data may also highlight the significance of an integrative measure of pro-apoptotic cytotoxicity for the
progression of heart failure beyond the assessment of
individual factors with a known capacity to induce cell
death. Serum levels of TNFa, which was among the first
serological factors to be recognised as deleterious during
the course of heart failure,9 were not significantly different in survivors compared to patients, who died during
follow-up. Only extreme elevations of TNFa levels (above
the 80%-tile or >3.75 pg/ml) were significantly associated
with an increased risk for mortality (P = 0.015 by univariate Cox regression analysis). The limited predictive value
of TNFa levels is in accordance with previous data, which
showed that blocking antibodies against TNFa did not
completely abolish apoptosis induction by the serum of
heart failure patients but only reduced it to a minor extent.5,6 Moreover, serum levels of C-reactive protein
(CRP), which is considered a surrogate marker of systemic pro-inflammatory activity, had no significant influence on mortality in our population. Taken together,
these data suggest that the combined action of more
than one cytotoxic factors, rather than TNFa, contributes to apoptosis induction in EC by the serum of patients
with heart failure.
A major limitation of the present study relates to the
rather small sample size of patients. However, by univariate analysis, the previously established predictive
parameters of HF mortality pro-BNP10 and creatinine
levels as well as advanced NYHA class and lower blood
pressure showed a highly significant association with
reduced survival, thus confirming that the patient population studied was indeed representative of HF characteristics. Despite the rather small sample size of the
patient cohort, the study size was sufficiently powered
to indicate the independent predictive value of the
pro-apoptotic activity in a multivariable regression
model including other established predictors of mortality
in HF. Moreover, ROC curve analysis disclosed the usefulness of measuring pro-apoptotic serum activity to predict
mortality in patients with HF with sensitivity and specificity similar to measuring serum pro-BNP levels, the prototypic prognostic serum marker in patients with HF.
Nevertheless, the development of a routinely accessible,
standardised measurement procedure for the assessment
of the pro-apoptotic serum activity based on the experimental design used in the present study will be necessary
to independently validate the prognostic relevance
within the scope of larger trials.
Another limitation of the present study is the rather
infrequent use of b-blockers among our patients, since
in the year 1998, when enrolment of patients into this
study began, the use of b-blockers in patients with advanced heart failure was less well-established. However,
we have previously reported that the b-blocker carvedilol, but not metoprolol protects EC against apoptosis in
vitro and in vivo in patients with heart failure.6 Thus, future determinations of the pro-apoptotic serum activity
are mandatory to confirm the survival impact of this
parameter at the current standard medication.
In conclusion, the pro-apoptotic serum activity as an
integrative measure of the combined cytotoxic serum
load represents a strong independent predictor of mortality in patients with HF, thus further underscoring the
importance of systemic inflammatory responses in the
pathophysiology of HF.
The pro-apoptotic serum activity
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cell apoptosis in congestive heart failure. Circulation
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8. Anker SD, Doehner W, Rauchhaus M et al. Uric acid and survival in
chronic heart failure: validation and application in metabolic,
1625
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tumor necrosis factor in severe chronic heart failure. N Engl J Med
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