(Circulation. 1997;95:1165-1168.)
© 1997 American Heart Association, Inc.
Articles |
Basic Fibroblast Growth Factor as a Biochemical Marker of Exercise-Induced Ischemia
the Division of Cardiology, Department of Medicine, Columbia-Presbyterian Medical Center, New York, NY.
Correspondence to Judah Weinberger, MD, Columbia-Presbyterian Hospital, 161 Fort Washington Ave, New York, NY 10032. E-mail jzw1{at}columbia.edu
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Abstract |
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Background Basic fibroblast growth factor (bFGF) is a mitogenic polypeptide that demonstrates enhanced expression and promotes angiogenesis in animal models of myocardial ischemia and infarction. Elevated levels of bFGF are present in the urine of humans with metastatic tumors, but its expression in human myocardial ischemia is unknown. Thus, we sought to determine whether urine levels of bFGF are altered by exercise-induced ischemia in humans.
Methods and Results Eighty-six patients underwent exercise thallium studies for evaluation of anginal symptoms. Urine levels of bFGF (corrected for urine creatinine) were determined by ELISA immediately before and between 2 and 4 hours after exercise. The change in urine bFGF level was compared between 43 patients with and 43 patients without exercise-induced ischemia. Patients with ischemia had an increase in urine bFGF compared with nonischemic patients (1052±245 versus -278±130 pg/g creatinine, P<.0001). Exercise, demographic, and clinical variables were assessed and analyzed for possible effect on bFGF response to exercise. By univariate analysis, a history of hypertension was negatively associated with a change in bFGF level (P<.05). No other variables were associated. By multivariate analysis, only bFGF response (P<.001) and age (P<.05) were independently related to exercise-induced ischemia.
Conclusions Significantly increased levels of bFGF are detected in the urine within hours of exercise-induced ischemia. Further studies are warranted to determine whether bFGF might serve as a useful circulating marker of myocardial ischemia in humans.
Key Words: growth substances • ischemia • peptides • prognosis
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Introduction |
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The diagnosis of acute myocardial infarction is facilitated by clinically available assays for creatine phosphokinase isoenzymes and more recently for troponin T and P-selectin, which may also be useful in identifying patients with unstable angina and threatened infarction.1 2 Presently, no such biochemical markers are readily available for the diagnosis of myocardial ischemia in patients with stable angina. The availability of such a biochemical marker would complement currently used clinical, ECG, and exercise imaging techniques in diagnosing cases with unclear presentation and could potentially obviate the need for more expensive testing in less equivocal presentations.
Local biochemical alterations in response to tissue ischemia or hypoxia include synthesis and secretion of mitogenic growth factors such as vascular endothelial growth factor and acidic and basic fibroblast growth factors (bFGF).3 4 These angiogenic proteins promote the proliferation of endothelial and vascular smooth muscle cells and formation of new blood vessels.5
bFGF, the most extensively studied of these polypeptides, is an 18- to 24-kD member of a family of heparin-binding polypeptides. It is widely distributed throughout the body, being found in many human neuroectodermal and mesodermal tissues, including the heart.6 It is located intracellularly and in the extracellular matrix of injured and cultured cells; it is often bound via heparin-like molecules.7 Its expression is enhanced in animal models of cerebral ischemia,8 9 skeletal muscle ischemia,10 and myocardial ischemia.11 12 It has recently been demonstrated that intracoronary administration of bFGF promotes angiogenesis and collateral formation in areas of ischemic and infarcted porcine and canine hearts.13 14 15 16
Serum levels of bFGF have been shown to be elevated in patients with a variety of tumors.17 18 19 Urine bFGF levels have been shown to be useful for the detection of human renal cell carcinomas.20 With the recent development of a highly sensitive urine ELISA, baseline levels can be accurately quantified in healthy control subjects, and elevated levels have been shown to correlate with the presence of multiple metastatic solid tumors, lymphoma, and leukemia.21
Thus, given the demonstrated induced expression of bFGF and its angiogenic role in animal models of myocardial ischemia, we sought to evaluate the usefulness of bFGF as a biochemical marker of exercise-induced ischemia.
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Methods |
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Study Patients
Over a 4-month period from March 1995 through July 1995, 86 outpatients (52 men and 34 women; mean±SD age, 60±10 years) were referred to the exercise laboratory for evaluation of chest pain with an exercise thallium study. Patients were excluded from analysis if they had a history of malignancy, peripheral vascular disease clinically (claudication) or by prior evaluation, coagulation disorders (including recent deep vein thrombosis or pulmonary embolus), or renal insufficiency or if they were unable to exercise. Patients were classified into two groups based on the presence or absence of ischemia by either ECG or scan criteria. Informed consent was obtained from all patients. The study protocol was approved by the Institutional Review Board of Columbia-Presbyterian Medical Center.
Exercise Thallium Test
Symptom-limited exercise testing was performed on a treadmill according to a standard Bruce protocol. Termination of exercise was at the discretion of the physician performing the test and was based on patient symptoms, ECG abnormalities, or the attainment of 
85% of maximal predicted heart rate; 3 mCi of 201Tl was injected 
40 seconds before termination of exercise. Planar scanning and SPECT imaging were performed after recovery, and redistribution scans were done at 4 hours. ECGs were analyzed for ischemic criteria, and scans were evaluated for the presence and distribution of fixed and reversible perfusion defects.
Urine bFGF Analysis
A baseline urine specimen was obtained just before exercise, and a postexercise specimen was obtained between 2 and 4 hours after exercise, before redistribution scanning. Levels of bFGF (pg/mL) in samples prepared from the urine were measured with the use of an ELISA assay (R&D Systems, Inc) that uses monoclonal antibodies raised against recombinant human bFGF. The detection limit of the immunoassay is 0.1 pg/mL. Urine creatinine levels (g/mL) were determined on baseline and postexercise samples with the use of a colorimetric assay (Sigma Diagnostics). The level of bFGF was corrected for variation in glomerular filtration rate as determined by urine creatinine (Cr) excretion and expressed as bFGF/Cr in units of pg/g. All sample assays were run in quadruplicate. The difference between baseline and postexercise bFGF level was determined by the formula 
bFGF=bFGFpost/Crpost-bFGFpre/Crpre.
Statistical Analysis
Results are expressed as mean±SEM. Statistical significance was defined as a two-tailed Student's t test value of P<.05. Unpaired Student's t test was used to compare continuous variables between two groups. 
2 analysis and Fisher's exact test were used to compare categorical variables between two groups. Single-factor ANOVA was used to compare continuous variables among multiple groups. Linear regression was performed to assess relations between two continuous variables. Multivariate analysis (backward stepwise multiple logistic regression) was performed on all continuous and categorical variables to assess independent relations, with ischemia as the independent variable. Statistical calculations were performed using the Excel Version 5.0 (Microsoft Corp) and SAS (SAS Institute) software packages.
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Results |
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Exercise Thallium
Forty-three patients (50%) had ischemia on the basis of either ECG criteria or thallium scan. Twenty-eight (33%) had ischemia on the basis of ECG, and 29 (34%) had ischemia on the basis of a reversible perfusion defect on thallium scan. Reversible perfusion defects were present in the right coronary and/or left circumflex artery distribution in 22 patients (76%) and in the left anterior descending artery distribution in 7 patients (24%) with ischemia. Fixed perfusion defects were present on 18 scans (21%): 11 (61%) in the right coronary or circumflex and 7 (39%) in the anterior descending artery distribution. Fifteen patients (17%) experienced chest pain during exercise. The distribution of bFGF levels in the two groups of patients is shown in the Figure
. On the basis of univariate analysis, the presence of ischemia was strongly associated with an increase in urine bFGF. The mean change in urine bFGF in response to exercise was -278±130 pg/g for patients without ischemia and 1052±245 pg/g for those with ischemia (P<.0001) (Table 1). This association remained significant when using ECG (P<.01) and scan (P=.001) criteria for ischemia separately. Among 25 patients with no increase in urine bFGF, only 2 had exercise-induced ischemia. Of the 61 patients with an increase in urine bFGF, 41 had exercise-induced ischemia. Notably, all 21 patients with an increase in bFGF of >700 pg/g had exercise-induced ischemia. Sixteen of these patients (76%) had diagnostic ECG changes during exercise.
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There was no relation between other exercise variables (heart ratexblood pressure product, Bruce stage achieved, or occurrence of chest pain) and change in bFGF. Ischemia in a right and/or circumflex coronary artery distribution was associated with a greater increase in bFGF than ischemia in a left anterior descending artery distribution (1498±441 versus 432±144 pg/g, P<.05). Scans with fixed defects were associated with a smaller increase in bFGF than scans with no fixed defects (93±234 versus 465±186 pg/g, P=.1), but this difference did not achieve statistical significance.
Baseline Clinical Variables
Patient demographics and clinical characteristics are represented in Table 2. On the basis of univariate analysis, there was a negative association between hypertension and exercise-induced urine bFGF response (67±166 pg/g with versus 810±275 pg/g without hypertension, P<.05) There was no relation between bFGF response and any other demographic or clinical variable. Although there was a trend toward higher baseline levels of bFGF in women (2040 versus 1489 pg/g, P=.1), men exhibited a greater bFGF increase in response to exercise, although it was not statistically significant (584±233 versus 85±157 pg/g, P=.08).
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Multivariate Analysis
To assess for independent predictors of ischemia, bFGF response and all exercise (eg, heart ratexblood pressure product, chest pain), demographic (eg, age, sex), clinical (eg, hypertension, heart failure, diabetes, myocardial infarction, hyperlipidemia, cerebrovascular accident, revascularization), and ECG (eg, left ventricular hypertrophy) variables were entered into a stepwise multiple logistic regression analysis (SAS). A greater bFGF response (continuous variable) was independently related to exercise-induced ischemia (P<.001). Increasing patient age was the only other variable independently related to ischemia (P<.05).
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Discussion |
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The results of the present study demonstrate that urine bFGF significantly increases in patients with exercise-induced ischemia. To our knowledge, a circulating biochemical marker of inducible ischemia has not previously been identified in humans. An increase in circulating serum levels of bFGF in patients undergoing coronary angioplasty compared with patients undergoing diagnostic catheterization has been reported.22 However, the administration of heparin, which is thought to release bFGF-like activity into the circulation through bFGF displacement from its extracellular matrix,23 and endothelial injury during coronary angioplasty, which might induce synthesis and release of bFGF,24 25 26 may have influenced this result. No patients in this study received heparin, and the effect of endothelial injury (venipuncture) would be minimized by the presence of a control (nonischemic) population.
The results of studies in animals suggest that even in the absence of ischemia, exercise may contribute to elevations of absolute amounts of circulating bFGF. Clarke and coworkers27 demonstrated that in vivo rat cardiac myofibers contain less bFGF after exercise-induced normal contraction caused by membrane disruption and that adrenergic stimulation of heart rate and contractility increased this bFGF release. Exercise and electrical stimulation have also been shown to induce expression of bFGF in skeletal muscle.28 29 In the present study, urine bFGF levels, normalized to a glomerular filtration rate surrogate (urine creatinine), were not related to the amount of effective exercise as reflected by Bruce stage achieved or heart ratexblood pressure product attained. We cannot discount the possibility that exercise-induced ischemia in noncardiac tissues contributed to the observed increase in bFGF, but the exclusion of patients with known peripheral vascular disease or claudication would minimize this effect.
Clinical and demographic characteristics did not significantly affect baseline bFGF levels, and the magnitude of change was not related to the baseline level. The trend toward higher baseline levels in women has been previously observed.20 Men had a trend to greater increase after exercise, which may be related to a greater myocardial mass and therefore quantitatively more ischemia, although this was not directly assessed. When analyzed separately, however, both men (P<.0001) and women (P<.05) had significant increases in bFGF when ischemic. If myocardial mass is a determinant, a history of hypertension might be expected to be associated with a greater bFGF response. Surprisingly, the opposite was observed. Patients with a history of hypertension had a smaller increase in bFGF than did those without hypertension. The effect of antihypertensive medications (which may also be anti-ischemic) was not assessed, but it may contribute to this observation, as adrenergic blockade and angiotensin inhibition may partially inhibit bFGF induction.30 Other factors likely contribute, since the presence of hypertrophy by ECG voltage criteria did not correlate with bFGF response and the effect of hypertension was no longer significant on multivariate analysis.
Ischemia in the distribution of the right coronary artery or left circumflex coronary artery was associated with a greater increase in bFGF than ischemia in the left anterior descending distribution. Although this observation might be unexpected given the respective amounts of myocardium supplied normally supplied by these arteries, the amount of viable myocardium subject to ischemia in this group of patients may have been smaller in the subgroup demonstrating left anterior descending ischemia, as this subgroup had a predominance of associated fixed defects by thallium scan (57% versus 18%, P=.07).
Study Limitations
First, measurement of bFGF in urine samples reflects variably elapsed time from exercise to sample collection and cannot be strictly regulated. The kinetics of bFGF excretion in urine are unknown. In addition, because only one sample was obtained after exercise, peak levels could not be assessed, and kinetics of excretion could not be estimated. Future studies that involve the careful collection of urine and multiple blood samples should be performed to address this limitation. Second, bFGF is present in multiple organs and tissues as well as skeletal muscle. Cardiac-specific isoforms have not been identified. Therefore, despite exclusion of potential confounding clinical variables and analysis for relations between variables, the possibility of significant noncardiac sources of ischemia-induced circulating bFGF cannot be excluded. This may have contributed to the overlap of data points when comparing ischemic with nonischemic patients in the Figure
and could decrease the predictive accuracy of the test, limiting its use as a screening tool in its present form. Third, confirmatory evidence of significant coronary disease by angiography was not available for analysis.
Conclusions
bFGF, an angiogenic peptide induced by ischemia, can be accurately measured in human urine. This study demonstrates that exercise-induced ischemia assessed by ECG and by thallium scintigraphy is associated with a significant increase in urine bFGF excretion, and this effect is independent of clinical, demographic, and exercise-related variables. Further confirmatory investigation must be performed to assess the usefulness of bFGF as a clinical marker of myocardial ischemia.
Received April 8, 1996; revision received October 16, 1996; accepted October 21, 1996.
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