1Department of Cardiology, University Hospital, Lille, France
2Department of Hematology, University Hospital, Lille, France
3Department of Biochemistry, University Hospital, Lille, France
4INSERM-ESPRI 2004EA 2693, Lille II University, Lille, France
5INSERM-U508, Institut Pasteur, Lille, France
Received 9 May 2005; revised 18 June 2005; accepted 7 July 2005; online publish-ahead-of-print 16 August 2005.
* Corresponding author: Service de Cardiologie B et Hémodynamique, Hôpital Cardiologique, Centre Hospitalier Régional, 59037 Lille Cedex, France. Tel: +33 3 20 44 50 08; fax: +33 3 20 44 51 30. E-mail address: ericvanbelle{at}aol.com
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Abstract |
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Methods and results In 488 consecutive patients undergoing elective coronary angioplasty, hsC-reactive protein, HGF, and VEGF levels were measured before heparin administration. The primary endpoint, a composite of death and myocardial infarction, occurred in 44 patients at a median follow-up of 14.9 months. At baseline, VEGF levels were related to C-reactive protein levels and inversely related to age; HGF levels were related to C-reactive protein levels, diabetes, and recent clinical instability. In the univariate analysis, HGF had a significant positive relationship (P=0.003) with the primary endpoint. A similar trend was observed for VEGF (P=0.11). The only three variables significantly associated with the primary endpoint in the multivariable Cox model were HGF (P=0.004), C-reactive protein (P=0.007), and diabetes (P=0.04).
Conclusion Our results demonstrate that in patients, without heparin pre-treatment, referred for PCR, a high serum level of HGF is an independent predictor of clinical events during follow-up and is correlated with other surrogate measures of the activity of atherosclerosis.
Key Words: Angioplasty Growth factors Diabetes mellitus C-reactive protein Clinical outcome
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Introduction |
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VEGF and HGF are heparin-binding growth factors, whose synthesis is induced by heparin administration in vivo.8 HGF levels are increased 550-fold after heparin administration in humans,9,10 and it is considered that heparin-released serum levels reflect the capability and the functional reserve of the organism to produce HGF.11
Conflicting results regarding the predictive value of angiogenic growth factors, specifically HGF, have recently been published. One study showed that, in patients with an acute coronary syndrome (ACS) treated with intravenous heparin, high serum levels of HGF were associated with a lower rate of subsequent adverse clinical events,12 whereas another showed that, in patients with renal failure, high levels of HGF were associated with carotid atherosclerosis and an increased rate of death and cardiovascular events.13
No study has addressed the predictive value of serum VEGF and HGF levels in patients with coronary artery disease (CAD) not treated by heparin. In consecutive patients referred for percutaneous coronary revascularization (PCR) who were not receiving heparin, we studied the relationship between VEGF and HGF levels and clinical outcome. We also examined the clinical and biological covariates of baseline serum levels of VEGF and HGF.
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Methods |
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Hypertension was defined as a known blood pressure >140/90 mmHg or use of antihypertensive drugs. Smoking was defined as acknowledged ceased/unceased smoking. Diabetes mellitus was defined as a fasting glucose 126 mg/dL, use of hypoglycaemic agents, or a history of physician-diagnosed diabetes mellitus. Obesity was defined as body mass index >30 kg/m2. Family history of premature CAD was defined as CAD in a male first-degree relative <55 years or CAD in a female first-degree relative <65 years.14
Angioplasty procedure
Balloon angioplasty and coronary stenting were performed according to the standard technique in our laboratory.15 All patients received aspirin 300 mg/day; a bolus dose of heparin (500010 000 IU) was administered just before PCR. The use of stents was left to the discretion of the operator. Patients who had conventional balloon angioplasty received aspirin alone, whereas patients who had coronary stent implantation received aspirin (325 mg) and clopidogrel (300 mg loading dose and 75 mg daily) for 4 weeks followed by aspirin alone.
Clinical follow-up
Long-term clinical follow-up beginning at the time of the index procedure was accomplished by a questionnaire completed by the patient or by telephone contact. Review of hospital records and contact with the referring physician enabled us to complete missing information. Information obtained included death, occurrence of MI, and target vessel revascularization (TVR) since the initial PCR procedure. In the peri-procedural period, MI was defined as the development of new pathological Q-waves or creatine kinase-MB (or total CK if CK-MB not available) more than three times normal. During the follow-up period, MI was defined as the occurrence of new pathological Q-waves, or onset of ischaemic symptoms or ischaemic ECG changes with total CK-MB (or total CK if CK-MB not available) more than two times normal.16 Angiographic follow-up was performed only in patients with recurrent angina and/or positive stress testing. TVR was defined as the percutaneous or bypass revascularization of any segment of the epicardial coronary artery containing the target lesion. The clinical follow-up was intended to be performed after 12 months. The median follow-up period was 14.9 months (interquartile: 12.018.5 months). The primary endpoint of our study was a composite of death and non-fatal MI.
Analysis of biological markers
Blood samples were drawn from the arterial sheath immediately after puncture under sterile conditions and before heparin administration. Plasma and serum samples were divided into aliquots and stored at 80°C until analysis. To determine C-reactive protein, HGF, and VEGF, blood was collected into a Vacutainer (Terumo, Venoject) tube with no additive as recommended by the manufacturer. HGF and VEGF were measured by enzyme-linked immunosorbent assay (R&D systems). C-reactive protein was measured by nephelometry (Dade-Behring). For each parameter, all samples were analysed in the same run on the same day. The detection limit for HGF was 40 pg/mL and 5 pg/mL for VEGF. Inter-assay variation in our laboratory was 8% for HGF and 7% for VEGF, and intra-assay variation was 4.5% for both techniques within the limits given by the manufacturer. Inter-assay and intra-assay variation for C-reactive protein were 7.28 and 2.65%, respectively, for low levels and 3.77 and 2%, respectively, for high levels within the limits given by the manufacturer.
Statistical analysis
Continuous variables with little to mild skewness were presented as mean±SD and continuous variables with skewed distribution were presented as median values (interquartile range). Discrete variables were presented as absolute number and percentages. The association between angiogenic growth factors (HGF, VEGF) and continuous variables were assessed using Spearman's correlation coefficient; for discrete variables, the association was assessed with a logistic regression analysis using the discrete variable as dependent variable and growth factors as independent variable. To study the relation between angiogenic growth factors and multiple categorical and continuous determinants, a multiple regression analysis was performed. Categorical independent variables were encoded as 0 (absent) or 1 (present). Continuous independent variables were incorporated without any change.
Event-free survival was estimated with the KaplanMeier method. Differences were tested with a log-rank test. For presentation purpose, angiogenic growth factors were incorporated as tertiles.
Hazard ratios were calculated using simple (univariate) and multiple (multivariable) Cox models. Three multivariable Cox regression models were established. In the first model, the relation was assessed adjusting on age and gender; in the second model, usual factors related to prognosis [diabetes, hypertension, smoking, obesity, family history of CAD, clinical symptoms, left ventricular ejection fraction (LVEF), C-reactive protein levels, LDL-cholesterol levels, use of statins] were added; and in the final model, medications associated with survival in univariate analysis [use of glycoprotein IIb/IIIa inhibitors or angiotensin-converting enzyme (ACE)-inhibitors] were added. Because these three models yield similar results, only values of the third model are presented. Because of skewness, log transformation of HGF, VEGF, and C-reactive protein, were used.
For each variable, the proportional hazards assumption was tested visually using KaplanMeier curves and by examining a plot of ln(ln(survival time)) against the ln(time). The assumption was tested statistically using the stphtest command in Strata, which is based on fitting a simple linear regression model with the Schoenfield residuals as the dependent variable and time as the independent variable and then testing whether the slope of the regression line is equal to zero. In addition, the proportional hazard was assessed and satisfied by including an interaction time-dependent term in the multivariable Cox regression analysis. All hypothesis were two-tailed with a 0.05 type I error rate.
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Results |
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Median C-reactive protein level was 2.60 mg/L (1.207.09), median VEGF level was 445 pg/mL (291676), and median HGF level was 1585 pg/mL (12872113).
Angiogenic growth factor levels and cardiovascular risk factors
Correlates of VEGF and HGF levels by univariate analysis as a function of baseline patient characteristics are presented in Table 1.
By multivariable analysis, higher VEGF levels were associated with higher C-reactive protein levels (P=0.0001) and were inversely correlated with age (P=0.02). Higher HGF levels were associated with diabetes mellitus (P=0.03), clinical instability (P=0.007), and with higher C-reactive protein levels (P=0.003).
Angiogenic growth factor levels and clinical outcome
Clinical follow-up was obtained in all patients at a median interval of 14.9 (12.018.6) months after inclusion. During follow-up, 24 patients died, 26 had an acute MI, and 49 had a TVR. A major coronary event (death or MI) occurred in 44 patients.
Higher HGF levels were associated with a higher risk of major coronary event during follow-up (death or MI, 2=8.62, HR=5.91 for one log increase, 95% CI=1.8119.25; P=0.003) (Table 2) and a similar trend was observed for VEGF levels (P=0.11, Table 2). A separate analysis was performed in patients with or without an ACS during the last 4 weeks. Among patients with a recent ACS, higher HGF levels were associated with a higher risk of major coronary event (
2=4.82, HR=4.23 for one log increase, 95% CI=1.1123.12; P=0.03). Similarly, among patients without a recent ACS, higher HGF levels were associated with a higher risk of major coronary event (
2=3.95, HR=6.63 for one log increase, 95% CI=1.0544.08; P=0.04).
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In multivariable analysis; with adjustment for age, gender, hypertension, smoking, obesity, family history of CAD, clinical symptoms, LDL cholesterol levels, LVEF, use of glycoprotein IIb/IIIa during the procedure, VEGF serum levels, and treatment with statins and ACE-inhibitors; three predictors of major coronary events were identified: diabetes mellitus (P=0.04), C-reactive protein levels (P=0.007), and HGF levels (P=0.004) (Table 3). Figure 1 illustrates the occurrence of events according to diabetic status, tertiles of C-reactive protein levels, and tertiles of HGF levels, and Figure 2 illustrates the combined prognostic value of tertiles of C-reactive protein and HGF levels.
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Effects of CAD and heparin on angiogenic growth factor levels
Additional patients had plasma and serum samples and were used for ancillary analyses. Forty-one consecutive patients with suspected stable angina but a normal angiogram (no stenosis >30% diameter by visual assessment) and no heparin treatment were used to confirm the effect of CAD on biological markers. Eighteen consecutive patients with an abnormal angiogram (at least one stenosis >50% diameter by visual assessment) but without recent (<30 days) unstable symptoms and treated with IV heparin for at least 24 h before blood sampling were used to confirm the effect of heparin on angiogenic growth factor levels.
In patients with stable angina and CAD from our study population (n=338), C-reactive protein [2.45 mg/L (0.995.93) vs. 1.86 mg/L (0.574.43)] and HGF [1538 pg/mL (12651928) vs. 402 pg/mL (230577)] levels were higher than that in 41 consecutive patients with suspected stable angina but a normal angiogram (P=0.03 and P=0.0001, respectively). There was no difference in VEGF [439 pg/mL (273660) vs. 402 pg/mL (230577)] levels between the two groups (P=0.16). Other major baseline characteristics were not different between the two groups (data not shown).
HGF and VEGF levels were also compared between a group of patients with CAD and stable angina pre-treated with heparin (n=18) with the subgroup of patients with stable angina in our study population (n=338). In patients receiving heparin, median HGF level [3622 pg/mL (17118701)] was 2.5-fold higher than in patients without heparin pre-treatment [1538 pg/mL (12651928), P=0.0001], whereas median VEGF level [481 pg/mL (428852)] was only slightly higher than in patients without heparin pre-treatment [439 pg/mL (273660), P=0.04]. Other major baseline characteristics were not different between the two groups (data not shown).
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Discussion |
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Clinical and biological correlates of VEGF and HGF levels
Very few studies, in relatively small cohorts, have investigated the correlates of VEGF and HGF in patients with cardiovascular disease.1719
In our study, VEGF levels were positively correlated with C-reactive protein levels and were inversely related to age. No relation was found between VEGF levels and other risk factors. The observed relation between VEGF and C-reactive protein levels is consistent with previous reports that demonstrated a similar relationship between VEGF levels and inflammatory markers, including C-reactive protein, in diverse clinical conditions.20 The observed relationship between VEGF and C-reactive protein could reflect the fact that both are regulated by interleukin 6.21
The inverse relationship between VEGF levels and age confirms previous observations in animals that VEGF expression decreases with age.22 A similar relationship was described in a recent study in 50 patients referred for coronary artery bypass surgery.23
As previously reported, HGF levels were higher in obese24 and smokers18 as well as in patients with CAD than in patients without CAD.5 Higher HGF levels were also observed in patients with a low ejection fraction. The latter finding could reflect induction of HGF production by myocardial injury.3
In multivariable analysis, HGF levels were related to C-reactive protein levels. As for VEGF, this relationship was previously reported in other clinical situations25 and was suggested to be related to induction by interleukin 6.26 Of importance, multivariable analysis demonstrated that HGF levels were related to two clinical situations, diabetes mellitus and a recent episode of unstable symptoms. It has been shown that, in patients with CAD, intra-arterial thrombus is more frequent in diabetic patients27 and can persist for weeks following an acute coronary event.28 Such arterial thrombus formation could increase circulating HGF as previously demonstrated in animal models8 and in humans.29 The mechanism suggested for this induction is thought to be related to the release of endogenous heparin during mast cell degranulation.8
The lack of relationship between HGF levels and thrombus, as detected by angiography, in the CAPTURE trial12 is not inconsistent with this hypothesis. Indeed, because of the poor sensitivity of angiography to detect thrombus, the study by Heeschen et al.12 was largely underpowered in that aspect. Although the frequency of thrombus in patients with ACS has been reported to be very high (6575%) using a very sensitive method such as angioscopy,27,30 it was detected in only 6% of cases by angiography in the CAPTURE trial.12
Predictive value of C-reactive protein level in our population
Elevated C-reactive protein has been identified as a strong predictor of prognosis in healthy individuals, in patients with stable or unstable angina, and in patients after an acute MI.31 More recent studies have demonstrated that C-reactive protein was also an independent predictor of events in patients referred for PCR.3234 Our study confirms these recent reports and extend their findings in a different patient population. Indeed, whereas in our study, a high proportion of patients were treated with coronary stents (91%), it was the case in only 30% of patients in the study by de Winter et al.32 Among studies in which the use of coronary stents was more liberal (>80%), it is also interesting to notice that, because patients with an acute MI or unstable symptoms requiring heparin treatment were excluded, our study confirms the results by Walter et al.33 and by Chan et al.34 in a population at lower risk where the median C-reactive protein level (2.6 mg/L) was much lower than in the populations studied by Walter et al. (6 mg/L) or Chan et al. (4.2 mg/L).
Predictive value of VEGF and HGF levels in our population
Because it is considered that angiogenic growth factors are involved in the vascular injury/repair process, it has been suggested that the serum levels of these growth factors could predict clinical outcome in patients with atherosclerosis.
Our study demonstrates that in patients with CAD referred for PCR, serum VEGF levels were a weak predictor of clinical events, whereas serum HGF levels were a strong and independent predictor of clinical events. Our findings on HGF are concordant with the results of a study performed in patients with renal insufficiency showing that patients with serum HGF levels >1475 pg/mL had a shorter survival.13
A recently published ancillary study of the CAPTURE trial reported the impact of VEGF and HGF levels on clinical outcome in patients with an ACS.12 This study differed significantly from our study with respect to patient selection. All patients in the CAPTURE trial had refractory unstable angina; in the present study, only one-third of the population had a history of an acute coronary event in the month preceding the PCR procedure. Similarly, in the CAPTURE trial, half of the population received glycoprotein IIb/IIIa inhibitors for 1824 h before coronary angioplasty, whereas no patient in our population was pre-treated with glycoprotein IIb/IIIa inhibitors before blood sampling and the start of the angioplasty procedure. Among other differences are the higher proportion of diabetic patients (29 vs. 10%) and patients treated with ACE-inhibitors (42 vs. 20%) in our study compared with the CAPTURE trial.
Another key difference between the two studies is the use of heparin. Indeed, blood samples were tested during heparin treatment in all patients in the CAPTURE trial, whereas they were tested in patients free of heparin in our study. The latter observation is of great importance because VEGF and HGF are heparin-binding growth factors and are known to be inducible by heparin administration in vivo.8 It has been reported that administration of unfractioned or low molecular weight heparin in humans can induce a 50-fold increase in serum levels of HGF after intravenous injection9,10 and a four- to five-fold increase following subcutaneous injection.9,10 This probably explains why the median HGF level was three-fold higher in the CAPTURE trial than in our study group or than in the study by Malatino et al.13 The 2.5-fold higher HGF level observed in an ancillary group of 18 patients pre-treated with heparin in our study is also consistent with this explanation.
Administration of heparin has already been employed as a test to evaluate the functional reserve of the organism to produce HGF, a growth factor involved in vascular and myocardial repair.11 This may explain the apparently conflicting observation in the CAPTURE study that stimulated HGF levels were associated with a protective effect on prognosis, whereas naturally circulating HGF, as measured in the present study and in the study by Malatino et al.,13 was a marker of poor prognosis. This prognostic value is probably related to the fact that naturally circulating HGF is produced in response to the atherothrombotic process and to myocardial damage as demonstrated by the analysis of correlates of HGF levels.
Finally, an alternative explanation could be that the study by Heeschen et al.12 and our study represent a continuum with low HGF levels reflecting the extent of the inflammatory process and thus events in a stable population, whereas high levels in an unstable population have therapeutic benefits (i.e. angiogenesis) and thus exert a protective effect.
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Study limitations |
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This was a single-centre study, and patient referral, PCR technique, and medical management may have influenced the results. However, the consecutive nature of the population and the exclusion of patients treated with heparin provide a unique insight into baseline levels of VEGF and HGF and of their biological and clinical correlates in patients with CAD. Finally, these characteristics in conjunction with the high rate of clinical follow-up (100%) allowed us to accurately evaluate the prognostic value of baseline levels of VEGF and HGF after PCR.
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Clinical implications |
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Footnotes |
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References |
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