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Serum basic fibroblast growth factor levels in patients with ischemic heart disease
International Journal of Cardiology 59 (1997) 133–138
Serum basic fibroblast growth factor levels in patients with ischemic heart disease David Hasdai a , Vivian Barak b , Eyal Leibovitz a , Itzhak Herz a , Samuel Sclarovsky a , Michael Eldar c , c, Mickey Scheinowitz * b
a Department of Cardiology, Beilinson Medical Center, Tel-Aviv University, Petah Tikva, Israel The Immunology and Tumor Diagnosis Laboratory, Department of Oncology, Hadassah Medical Center, Hebrew University, Jerusalem, Israel c The Neufeld Cardiac Research Institute, Tel-Aviv University, Tel-Hashomer, Israel
Received 19 November 1996; accepted 19 December 1996
Abstract Background: Being a potent promoter of endothelial and smooth muscle cell proliferation, basic fibroblast growth factor (bFGF) is presumed to play a key role in coronary collateral development and atherogenesis. Purpose: To characterize serum bFGF levels in patients with ischemic heart disease. Methods: The study population consisted of patients with angina (n533) and after uncomplicated myocardial infarction (n512). The number of significantly stenosed ($50%) vessels and angiographic coronary collateral score were noted. Blood was drawn immediately prior to elective coronary angiography in study patients for bFGF levels. Twenty healthy, age-matched subjects served as control for serum bFGF. Results: Serum bFGF levels were undetectable in all 20 control subjects, but were detectable in 15 / 33 (45%) patients with angina and 3 / 12 (25%) post-infarction patients, respectively (P50.002). Serum bFGF levels were detectable in 13 / 23 (57%) patients with 0- or 1-vessel disease, as compared with 5 / 22 (23%) patients with 2- or 3-vessel disease (P,0.05). Detectable serum bFGF levels were not in correlation with coronary collateral score (P51). Conclusions: Serum levels of bFGF are elevated in patients with ischemic heart disease, particularly in those with minimal coronary artery disease. We postulate that detectable serum bFGF levels reflect active atherogenesis rather than myocardial collateral development. 1997 Elsevier Science Ireland Ltd. Keywords: Basic fibroblast growth factor; Ischemic heart disease; Atherosclerosis; Collateral circulation
1. Introduction Basic fibroblast growth factor (bFGF) is a 18-kDa molecule with widespread tissue distribution, stored primarily in the endothelial basement membrane [1]. Being a potent mitogen of cells of mesenchymal origin, it promotes proliferation of endothelial and smooth muscle cells [1]. Angiogenesis is the key to coronary collateral *Corresponding author, Neufeld Cardiac Research Institute, Sheba Medical Center, Tel Hashomer, Israel 52621. Tel.: 1972 3 5302614 / 5342278; fax: 1972 3 5351139.
development in response to myocardial ischemia [2]. Exogenously-administered bFGF has been shown to induce myocardial angiogenesis in different experimental models of myocardial ischemia / infarction [3–7], thus increasing coronary collateral flow [3,5– 7]. Angiogenesis is also a fundamental process in tumor growth and metastasis [8]. Using an immunoassay for bFGF [9], increased serum and urine levels of bFGF have been reported in patients with different malignancies [10–12], supposedly due to bFGF production by tumor cells [13]. As a common process to tumor metastasis and myocardial ischemia, one
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could postulate that angiogenesis in response to myocardial ischemia would result in elevated systemic levels of bFGF. Atherogenesis is a complex process entailing smooth muscle proliferation and angiogenesis within the vessel wall [14]. Several workers have previously shown that bFGF might be an important participant in atherogenesis [15–19]. As atherosclerosis is a systemic process with widespread involvement, serum bFGF might be elevated in patients with ischemic heart disease and coronary artery atherosclerosis. The purpose of the present study was to examine whether serum levels of bFGF are detectable in patients with ischemic heart disease, and to evaluate a possible correlation between serum bFGF levels and extent of coronary artery disease and coronary collateral circulation.
2. Methods
2.1. Study population The study protocol was approved by our institutional review board. All patients gave informed consent after the purpose of the study was explained to them. The study population consisted of 33 patients undergoing elective coronary angiography due to presence of typical anginal pain and 12 patients recovering from uncomplicated acute myocardial infarction (7.261.9 days post-infarction). Patients were not required to undergo noninvasive evaluation for ischemia prior to coronary angiography; the decision to refer patients to coronary angiography was at the discretion of the attending physician. In all patients experiencing chest pain, the cause was presumed to be cardiac ischemia, after other causes were ruled out. Patients with unstable angina underwent coronary angiography after their symptoms had subsided with pharmacological therapy (asymptomatic for 2–3 days). Heparin administration was stopped at least 6 h prior to blood tests; all other drugs were administered as usual. Patients with severe heart failure (NYHA class III–IV), neoplastic, immunologic, infectious, or inflammatory disease were excluded.
2.2. Angiographic data The degree of coronary artery stenosis in coronary angiography (Judkin’s technique) was determined in at least two projections by two independent investigators. Stenosis was considered significant if $50% of luminal diameter was occluded. The study group was divided into three subgroups: (I) Stable (n56) and unstable (n54) angina without significant coronary artery stenosis, (II) stable (n59) and unstable (n514) angina with significant coronary artery stenosis, and (III) post-infarction (n512). The presence of collaterals was scored according to Rentrop’s classification [20]. Collateral vessels were considered absent when graded 0 or 1 and present when graded 2 or 3. In case of more than one artery receiving collateral circulation, the score given to the artery with the richest collateral circulation was registered.
2.3. bFGF assay All blood samples were taken from patients in the fasting state (at least 6 h). Immediately prior to coronary angiography, venous blood was drawn and centrifuged for 5 min at 2000 rev. / min, and the serum was separated and stored at 2208C for ensuing analysis. Twenty healthy, age-matched volunteers served as control for serum bFGF. Serum bFGF levels were measured using a solid phase ELISA kit (R & D systems, Minneapolis, MN), as previously described [9]. The detection limit of the assay is 1 pg / ml.
2.4. Statistics Mean6SD was calculated for continuous variables (age and serum bFGF levels), and absolute frequencies were measured for discrete variables. In case of continuous variables differences between groups were examined for statistical significance using one-way analysis of variance (ANOVA). The chi-square test was applied to compare the statistical significance between discrete variables. In cases of small numbers of patients in each category, Fisher’s exact test was performed. All tests were 2-tailed, and P-values #0.05 were considered statistically significant.
D. Hasdai et al. / International Journal of Cardiology 59 (1997) 133 – 138
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3. Results The demographic and clinical characteristics of the three subgroups comprising the study population are presented in Table 1. Patients with angina and minimal coronary artery disease tended to be younger, but there was no difference between the three subgroups in gender or risk factors. There were subtle differences in drug therapy between the subgroups. Among patients in subgroups II and III, patients in subgroup II had a significantly higher history of previous myocardial infarction, but no significant difference in history of previous angioplasty. The number of vessels involved was similar in subgroups II and III. Fig. 1 depicts the distribution of serum bFGF levels in the respective subgroups and in healthy control subjects. Whereas serum bFGF levels were undetectable in all 20 control subjects, it was detectable in six (60%), nine (39%), and three (25%) patients belonging to subgroups I, II, and III, respectively (P50.004). In subgroups I and II, there was no difference between patients with stable or unstable angina in terms of detectable bFGF levels (Group I:
Fig. 1. Serum bFGF levels in normal healthy controls, patients with angina without significant coronary artery stenosis (I), patients with angina with significant coronary artery stenosis (II), and patients after myocardial infarction (III).
2 / 6 (33%) patients with stable vs. 4 / 4 (100%) patients with unstable angina, P5NS. Group II: 5 / 9 (56%) patients with stable vs. 7 / 14 (50%) patients with unstable angina, P5NS). In all, serum bFGF levels were detectable in 15 / 33 (45%) patients with angina (regardless of extent of coronary artery disease), and 3 / 12 (25%) post-infarction patients, respectively (P50.002 compared with control).
Table 1 Demographic, clinical and angiographic characteristics of patients Characteristic
No. of patients Age (years) Sex (M / F) Prior myocardial infarction Prior angioplasty Diabetes mellitus Hypertension Hypercholesterolemia Drug therapy Aspirin Nitrates b -Blockers Calcium-channel blockers Diuretics Ace-inhibitors** Coronary artery disease One-vessel Two-vessel Three-vessel
Group
P-value
I
II
III
10 56614 8 / 2 (80% / 20%) 0 (0%) 0 (0%) 2 (20%) 5 (50%) 4 (40%)
23 6669.8 17 / 6 (74% / 26%) 16 (70%) 5 (22%) 7 (30%) 10 (43%) 14 (61%)
12 65611 8 / 4 (67% / 33%) 3 (23%) 1 (7%) 2 (15%) 8 (62%) 5 (38%)
7 4 1 6 2 2
20 15 4 15 6 6
12 2 4 2 1 5
(70%) (40%) (10%) (60%) (20%) (20%)
0 (0%) 0 (0%) 0 (0%)
(87%) (65%) (17%) (65%) (26%) (26%)
6 (26%) 6 (26%) 11 (48%)
(100%) (17%) (33%) (17%) (8%) (42%)
0.054 NS 0.03* NS* NS NS NS ,0.05 ,0.05 NS 0.05 NS NS NS*
5 (42%) 2 (16%) 5 (42%)
Characteristics of patients with angina without significant coronary artery stenosis (I), patients with angina with significant coronary artery stenosis (II), and patients after myocardial infarction (III) are shown. *P-value for comparison between subgroups II and III. P-value for coronary artery disease represents differences in distribution of one-, two-, and three-vessel disease in both groups. **ACE, angiotensin converting enzyme.
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Elevated serum bFGF levels were elevated in patients with less extensive coronary artery disease (elevated in 60%, 55%, 38%, and 23% of patients with 0-, 1-, 2-, and 3-vessel disease, respectively, P50.13). In fact, serum bFGF levels were detectable in 13 / 23 (57%) patients with 0- or 1-vessel disease, as compared with 5 / 22 (23%) patients with 2- or 3-vessel disease (P,0.05). Detectable serum bFGF levels were not indicative of the presence of coronary collateral circulation (detectable in 32% of patients without vs. 33% with collateral circulation, P51).
4. Discussion
4.1. Main findings We found that serum levels of bFGF are elevated in patients with ischemic heart disease. Elevated serum bFGF levels are found more commonly in patients with minimal coronary artery disease, and are not indicative of status of coronary collateral circulation. Among patients recovering from myocardial infarction, in which bFGF is expected to participate not only in formation of collateral circulation, but also in wound repair and healing [4], bFGF is detectable in only a minority of patients.
4.2. Possible explanations As for metastatic malignancies, the source for the detectable systemic levels of bFGF is intriguing. An animal study designed to distinguish between tumorderived and host-derived bFGF concluded that systemic bFGF most likely originates from tumor cells and not from host cells [13]. Our study, as well as a previous one [21], shows that serum bFGF may be elevated in the absence of malignancy, suggesting that host cells can either produce or secrete bFGF in excess under certain conditions. There are several mechanisms which may explain increased bFGF production and / or secretion in ischemic heart disease. In experimental models of regional myocardial ischemia, growth factors such as acidic fibroblast growth factor and vascular endothelial growth factor are activated in the ischemic region of the myocardium, but not in normal regions [22,23]. Increased
expression of myocardial bFGF during ischemia has been shown in vivo by some [24], but not by other investigators [22]. Thus, although it is tempting to assume that the source for the detectable levels of bFGF found in our study population was the result of increased production and / or secretion from the ischemic myocardium, firm evidence to support this assumption is still lacking. In the course of angiogenesis, the vascular basement membrane is disrupted, resulting in the release of bFGF [2]. It could therefore be suggested that bFGF are detectable in patients in whom coronary collaterals are developing. In our study, detectable serum bFGF levels were not indicative of the presence of collateral circulation. We also found that serum bFGF levels were particularly detectable in patients with minimal coronary artery stenosis and in patients with less extensive coronary artery disease. These findings do not contradict the possibility that serum bFGF levels are increased due to myocardial angiogenesis and coronary collateral development, because collateral circulation may develop in the absence of obstructive coronary artery disease [25]. Moreover, since coronary angiography was performed at one point of time, we cannot determine the dynamics of collateral circulation development, and thus might have obtained samples from patients in different stages of collateral circulation development. Nevertheless, these data suggest that detectable serum bFGF levels may reflect processes unrelated to myocardial angiogenesis and development of collateral circulation. Detectable serum levels of bFGF in patients with ischemic heart disease may reflect active atherogenesis. Growth factors, including bFGF, are presumed to orchestrate the chain of events culminating in the formation of the atherosclerotic plaque [14]. Fibroblast growth factors (acidic and basic) have been implicated in the pathogenesis and disruption of atherosclerotic plaques [15–19]. Indeed, increased expression of bFGF, and more so of acidic fibroblast growth factor, have been exhibited in human atheromatous tissue [15–18]. Chen et al. [15] found bFGF in human coronary arteries with proliferative lesions, but not in fibrous plaques. Moreover, it is possible to attenuate smooth muscle proliferation in these lesions by adding a neutralizing antibody to bFGF or bFGF antisense oligonucleotides [15]. Simi-
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larly, Liau et al. [16] reported decreased mRNA for bFGF (and increased mRNA for acidic fibroblast growth factor) in advanced atheromatous lesions in hypercholesterolemic pigs, and increased mRNA for bFGF (and decreased mRNA for acidic fibroblast growth factor) in normal-appearing regions adjacent to these lesions. Therefore, it seems that bFGF plays a role in the early stages of plaque formation characterized by intense smooth muscle proliferation, and not in advanced atherosclerosis (fibrous plaque).
4.3. Limitations Serum bFGF levels were determined only once in our patients. It is possible that serum bFGF levels fluctuate, and thus we might have underestimated the prevalence of elevated bFGF levels among ischemic patients. A rise in bFGF levels may reflect decreased clearance or uptake, and / or increased production or secretion. In our study, we could not determine which of these mechanisms was responsible for the elevation in serum bFGF levels. Three, serum bFGF levels might not reflect tissue levels of bFGF. The correlation between local and systemic levels of bFGF remains to be determined in future studies. Four, differences in concomitant drug therapy may have influenced serum bFGF levels. To our knowledge, there are little data regarding the effect of antiischemic therapy on circulating bFGF levels.
References [1] Folkman J, Klagsburn M. Angiogenic factors. Science 1987; 235: 442–447. [2] Schaper W, Sharma HS, Quinkler W, Markert T, Wunsch M, Schaper J. Molecular biologic concepts of coronary anastomoses. J Am Coll Cardiol 1990; 15: 513–518. [3] Yanagisawa-Miwa Y, Uchida Y, Nakamura F, Tomaru T, Kido H, Kamijo T, Sugimoto T, Kaji K, Utsuyama M, Kurashima C, Ito H. Salvage of infarcted myocardium by angiogenic action of basic fibroblast growth factor. Science 1992; 257: 1401–1403. [4] Battler A, Scheinowitz M, Bor A, Hasdai D, Vered Z, Di Segni E, Varda-Bloom N, Nass D, Engelberg S, Eldar M, Belkin M, Savion N. Intracoronary injection of basic fibroblast growth factor enhances angiogenesis in infarcted swine myocardium. J Am Coll Cardiol 1993; 22: 2001–2006. [5] Harada K, Grossman W, Friedman M, Edelman ER, Prasad PV, Keighley CS, Manning WJ, Selike FW, Simons M. Basic fibroblast growth improves myocardial function in chronically ischemic porcine hearts. J Clin Invest 1994; 94: 623–630.
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[6] Unger EF, Banai S, Shou M, Lazarous DF, Jaclitsch MT, Scheinowitz M, Correa R, Klingbeil C, Epstein SE. Basic fibroblast growth factor enhances myocardial collateral blood flow in a canine model. Am J Physiol 1994; 266: H1588–H1595. [7] Lazarous DF, Scheinowitz M, Shou M, Hodge E, Rajanayagam MAS, Hunsberger S, Robinson WG Jr, Stiber JA, Correa R, Epstein SE, Unger EF. Effects of chronic systemic administration of basic fibroblast growth factor on collateral development in the canine heart. Circulation 1995; 91: 145–153. [8] Folkman J, Watson K, Ingber D, Hanahan D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature (Lond.) 1989; 339: 58–61. [9] Watanabe H, Hori A, Seno M, Kozai Y, Igarashi K, Ichimori Y, Kondo K. A sensitive enzyme immunoassay for human basic fibroblast growth factor. Biochem Biophys Res Commun 1991; 175: 229–235. [10] Fujimoto K, Ichimori Y, Kakizoe T, Okajima E, Sakamoto H, Sugimura T, Terada M. Increased serum levels of basic fibroblast growth factor in patients with renal cell carcinoma. Biochem Biophys Res Commun 1991; 180: 386–392. [11] Nguyen M, Watanabe H, Budson AE, Richie JP, Folkman J. Elevated plasma levels of the angiogenic peptide basic fibroblast growth factor in urine of bladder cancer patients. J Natl Cancer Inst 1993; 85: 241–242. [12] Nguyen M, Watanabe H, Budson AE, Richie JP, Hayes DF, Folkman J. Elevated levels of an angiogenic peptide, basic fibroblast growth factor, in the urine of patients with a wide spectrum of cancers. J Natl Cancer Inst 1994; 86: 359–361. [13] Soutter AD, Nguyen M, Watanabe H, Folkman J. Basic fibroblast growth factor secreted by an animal tumor is detectable in urine. Cancer Res 1993; 53: 5297–5299. [14] Ross R. The pathogenesis of atherosclerosis — an update. N Engl J Med 1986; 314: 488–500. [15] Chen CH, Nguyen HH, Henry PD, Gotto AM. Inhibition of atherosclerotic human coronary smooth muscle cell proliferation by blocking endogenous basic fibroblast growth factor [abstract]. Circulation 1994; 90: I-511. [16] Liau G, Winkles JA, Cannon MS, Kuo L, Chilian WM. Dietaryinduced atherosclerotic lesions have increased levels of acidic FGF mRNA and altered cytoskeletal and extracellular matrix mRNA expression. J Vasc Res 1993; 30: 327–332. [17] Flugelman FY, Virmani R, Correa R, Yu ZX, Farb A, Leon MB, Elami A, Fu YM, Casscells W, Epstein SE. Smooth muscle cell abundance and fibroblast growth factors in coronary lesions of patients with nonfatal unstable angina. A clue to the mechanism of transformation from the stable to the unstable clinical state. Circulation 1993; 88: 2493–2500. [18] Hughes SE, Crossman D, Hall Pa. Expression of basic and acidic fibroblast growth factors and their receptor in normal and atherosclerotic human arteries. Cardiovasc Res 1993; 27: 1214–1219. [19] Brogi E, Winkles JA, Underwood R, Clinton SK, Alberts GF, Libby P. Distinct patterns of expression of fibroblast growth factors and their receptors in human atheroma and nonatherosclerotic arteries. Association of acidic FGF with plaque microvessels and macrophages. J Clin Invest 1993; 92: 2408–2418. [20] Rentrop KP, Cohen M, Blanke H, Phillips R. Changes in collateral filling immediately following controlled coronary artery occlusion by an angioplasty balloon in man. J Am Coll Cardiol 1985; 5: 587–592. [21] D’Amore PA, Brown RH Jr, Ku PT, Hoffman EP, Watanabe H, Arahata K, Ishihara T, Folkman J. Elevated basic fibroblast growth factor in the serum of patients with Duchene muscular dystrophy. Ann Neurol 1994; 35: 362–365.
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[22] Bernotat-Danielowski S, Sharma HS, Schott RJ, Schaper W. Generation and localisation of monoclonal antibodies against fibroblast growth factors in ischaemic collateralized porcine myocardium. Cardiovasc Res 1993; 27: 1220–1228. [23] Sharma HS, Schaper W. The role of growth factors during development of a collateral circulation in the porcine heart. In: Schaper W, Schaper J, editors. Collateral circulation: heart, brain, kidney, limbs. Norwell, MA: Kluwer Academic Publishers, 1993; 123–147.
[24] Cohen MV, Vernon J, Yaghdjian V, Hatcher VB. Longitudinal changes in myocardial basic fibroblast growth factor (FGF-2) activity following coronary artery ligation in the dog. J Mol Cell Cardiol 1994; 26: 683–690. [25] Lisenmeyer GJ, Schneider JF. Angiographically visible intracoronary collateral circulation in the absence of obstructive coronary artery disease. Am J Cardiol 1984; 53: 954–956.
Serum basic fibroblast growth factor levels in patients with ischemic heart disease David Hasdai a , Vivian Barak b , Eyal Leibovitz a , Itzhak Herz a , Samuel Sclarovsky a , Michael Eldar c , c, Mickey Scheinowitz * b
a Department of Cardiology, Beilinson Medical Center, Tel-Aviv University, Petah Tikva, Israel The Immunology and Tumor Diagnosis Laboratory, Department of Oncology, Hadassah Medical Center, Hebrew University, Jerusalem, Israel c The Neufeld Cardiac Research Institute, Tel-Aviv University, Tel-Hashomer, Israel
Received 19 November 1996; accepted 19 December 1996
Abstract Background: Being a potent promoter of endothelial and smooth muscle cell proliferation, basic fibroblast growth factor (bFGF) is presumed to play a key role in coronary collateral development and atherogenesis. Purpose: To characterize serum bFGF levels in patients with ischemic heart disease. Methods: The study population consisted of patients with angina (n533) and after uncomplicated myocardial infarction (n512). The number of significantly stenosed ($50%) vessels and angiographic coronary collateral score were noted. Blood was drawn immediately prior to elective coronary angiography in study patients for bFGF levels. Twenty healthy, age-matched subjects served as control for serum bFGF. Results: Serum bFGF levels were undetectable in all 20 control subjects, but were detectable in 15 / 33 (45%) patients with angina and 3 / 12 (25%) post-infarction patients, respectively (P50.002). Serum bFGF levels were detectable in 13 / 23 (57%) patients with 0- or 1-vessel disease, as compared with 5 / 22 (23%) patients with 2- or 3-vessel disease (P,0.05). Detectable serum bFGF levels were not in correlation with coronary collateral score (P51). Conclusions: Serum levels of bFGF are elevated in patients with ischemic heart disease, particularly in those with minimal coronary artery disease. We postulate that detectable serum bFGF levels reflect active atherogenesis rather than myocardial collateral development. 1997 Elsevier Science Ireland Ltd. Keywords: Basic fibroblast growth factor; Ischemic heart disease; Atherosclerosis; Collateral circulation
1. Introduction Basic fibroblast growth factor (bFGF) is a 18-kDa molecule with widespread tissue distribution, stored primarily in the endothelial basement membrane [1]. Being a potent mitogen of cells of mesenchymal origin, it promotes proliferation of endothelial and smooth muscle cells [1]. Angiogenesis is the key to coronary collateral *Corresponding author, Neufeld Cardiac Research Institute, Sheba Medical Center, Tel Hashomer, Israel 52621. Tel.: 1972 3 5302614 / 5342278; fax: 1972 3 5351139.
development in response to myocardial ischemia [2]. Exogenously-administered bFGF has been shown to induce myocardial angiogenesis in different experimental models of myocardial ischemia / infarction [3–7], thus increasing coronary collateral flow [3,5– 7]. Angiogenesis is also a fundamental process in tumor growth and metastasis [8]. Using an immunoassay for bFGF [9], increased serum and urine levels of bFGF have been reported in patients with different malignancies [10–12], supposedly due to bFGF production by tumor cells [13]. As a common process to tumor metastasis and myocardial ischemia, one
0167-5273 / 97 / $17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0167-5273( 97 )02921-5
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could postulate that angiogenesis in response to myocardial ischemia would result in elevated systemic levels of bFGF. Atherogenesis is a complex process entailing smooth muscle proliferation and angiogenesis within the vessel wall [14]. Several workers have previously shown that bFGF might be an important participant in atherogenesis [15–19]. As atherosclerosis is a systemic process with widespread involvement, serum bFGF might be elevated in patients with ischemic heart disease and coronary artery atherosclerosis. The purpose of the present study was to examine whether serum levels of bFGF are detectable in patients with ischemic heart disease, and to evaluate a possible correlation between serum bFGF levels and extent of coronary artery disease and coronary collateral circulation.
2. Methods
2.1. Study population The study protocol was approved by our institutional review board. All patients gave informed consent after the purpose of the study was explained to them. The study population consisted of 33 patients undergoing elective coronary angiography due to presence of typical anginal pain and 12 patients recovering from uncomplicated acute myocardial infarction (7.261.9 days post-infarction). Patients were not required to undergo noninvasive evaluation for ischemia prior to coronary angiography; the decision to refer patients to coronary angiography was at the discretion of the attending physician. In all patients experiencing chest pain, the cause was presumed to be cardiac ischemia, after other causes were ruled out. Patients with unstable angina underwent coronary angiography after their symptoms had subsided with pharmacological therapy (asymptomatic for 2–3 days). Heparin administration was stopped at least 6 h prior to blood tests; all other drugs were administered as usual. Patients with severe heart failure (NYHA class III–IV), neoplastic, immunologic, infectious, or inflammatory disease were excluded.
2.2. Angiographic data The degree of coronary artery stenosis in coronary angiography (Judkin’s technique) was determined in at least two projections by two independent investigators. Stenosis was considered significant if $50% of luminal diameter was occluded. The study group was divided into three subgroups: (I) Stable (n56) and unstable (n54) angina without significant coronary artery stenosis, (II) stable (n59) and unstable (n514) angina with significant coronary artery stenosis, and (III) post-infarction (n512). The presence of collaterals was scored according to Rentrop’s classification [20]. Collateral vessels were considered absent when graded 0 or 1 and present when graded 2 or 3. In case of more than one artery receiving collateral circulation, the score given to the artery with the richest collateral circulation was registered.
2.3. bFGF assay All blood samples were taken from patients in the fasting state (at least 6 h). Immediately prior to coronary angiography, venous blood was drawn and centrifuged for 5 min at 2000 rev. / min, and the serum was separated and stored at 2208C for ensuing analysis. Twenty healthy, age-matched volunteers served as control for serum bFGF. Serum bFGF levels were measured using a solid phase ELISA kit (R & D systems, Minneapolis, MN), as previously described [9]. The detection limit of the assay is 1 pg / ml.
2.4. Statistics Mean6SD was calculated for continuous variables (age and serum bFGF levels), and absolute frequencies were measured for discrete variables. In case of continuous variables differences between groups were examined for statistical significance using one-way analysis of variance (ANOVA). The chi-square test was applied to compare the statistical significance between discrete variables. In cases of small numbers of patients in each category, Fisher’s exact test was performed. All tests were 2-tailed, and P-values #0.05 were considered statistically significant.
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3. Results The demographic and clinical characteristics of the three subgroups comprising the study population are presented in Table 1. Patients with angina and minimal coronary artery disease tended to be younger, but there was no difference between the three subgroups in gender or risk factors. There were subtle differences in drug therapy between the subgroups. Among patients in subgroups II and III, patients in subgroup II had a significantly higher history of previous myocardial infarction, but no significant difference in history of previous angioplasty. The number of vessels involved was similar in subgroups II and III. Fig. 1 depicts the distribution of serum bFGF levels in the respective subgroups and in healthy control subjects. Whereas serum bFGF levels were undetectable in all 20 control subjects, it was detectable in six (60%), nine (39%), and three (25%) patients belonging to subgroups I, II, and III, respectively (P50.004). In subgroups I and II, there was no difference between patients with stable or unstable angina in terms of detectable bFGF levels (Group I:
Fig. 1. Serum bFGF levels in normal healthy controls, patients with angina without significant coronary artery stenosis (I), patients with angina with significant coronary artery stenosis (II), and patients after myocardial infarction (III).
2 / 6 (33%) patients with stable vs. 4 / 4 (100%) patients with unstable angina, P5NS. Group II: 5 / 9 (56%) patients with stable vs. 7 / 14 (50%) patients with unstable angina, P5NS). In all, serum bFGF levels were detectable in 15 / 33 (45%) patients with angina (regardless of extent of coronary artery disease), and 3 / 12 (25%) post-infarction patients, respectively (P50.002 compared with control).
Table 1 Demographic, clinical and angiographic characteristics of patients Characteristic
No. of patients Age (years) Sex (M / F) Prior myocardial infarction Prior angioplasty Diabetes mellitus Hypertension Hypercholesterolemia Drug therapy Aspirin Nitrates b -Blockers Calcium-channel blockers Diuretics Ace-inhibitors** Coronary artery disease One-vessel Two-vessel Three-vessel
Group
P-value
I
II
III
10 56614 8 / 2 (80% / 20%) 0 (0%) 0 (0%) 2 (20%) 5 (50%) 4 (40%)
23 6669.8 17 / 6 (74% / 26%) 16 (70%) 5 (22%) 7 (30%) 10 (43%) 14 (61%)
12 65611 8 / 4 (67% / 33%) 3 (23%) 1 (7%) 2 (15%) 8 (62%) 5 (38%)
7 4 1 6 2 2
20 15 4 15 6 6
12 2 4 2 1 5
(70%) (40%) (10%) (60%) (20%) (20%)
0 (0%) 0 (0%) 0 (0%)
(87%) (65%) (17%) (65%) (26%) (26%)
6 (26%) 6 (26%) 11 (48%)
(100%) (17%) (33%) (17%) (8%) (42%)
0.054 NS 0.03* NS* NS NS NS ,0.05 ,0.05 NS 0.05 NS NS NS*
5 (42%) 2 (16%) 5 (42%)
Characteristics of patients with angina without significant coronary artery stenosis (I), patients with angina with significant coronary artery stenosis (II), and patients after myocardial infarction (III) are shown. *P-value for comparison between subgroups II and III. P-value for coronary artery disease represents differences in distribution of one-, two-, and three-vessel disease in both groups. **ACE, angiotensin converting enzyme.
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Elevated serum bFGF levels were elevated in patients with less extensive coronary artery disease (elevated in 60%, 55%, 38%, and 23% of patients with 0-, 1-, 2-, and 3-vessel disease, respectively, P50.13). In fact, serum bFGF levels were detectable in 13 / 23 (57%) patients with 0- or 1-vessel disease, as compared with 5 / 22 (23%) patients with 2- or 3-vessel disease (P,0.05). Detectable serum bFGF levels were not indicative of the presence of coronary collateral circulation (detectable in 32% of patients without vs. 33% with collateral circulation, P51).
4. Discussion
4.1. Main findings We found that serum levels of bFGF are elevated in patients with ischemic heart disease. Elevated serum bFGF levels are found more commonly in patients with minimal coronary artery disease, and are not indicative of status of coronary collateral circulation. Among patients recovering from myocardial infarction, in which bFGF is expected to participate not only in formation of collateral circulation, but also in wound repair and healing [4], bFGF is detectable in only a minority of patients.
4.2. Possible explanations As for metastatic malignancies, the source for the detectable systemic levels of bFGF is intriguing. An animal study designed to distinguish between tumorderived and host-derived bFGF concluded that systemic bFGF most likely originates from tumor cells and not from host cells [13]. Our study, as well as a previous one [21], shows that serum bFGF may be elevated in the absence of malignancy, suggesting that host cells can either produce or secrete bFGF in excess under certain conditions. There are several mechanisms which may explain increased bFGF production and / or secretion in ischemic heart disease. In experimental models of regional myocardial ischemia, growth factors such as acidic fibroblast growth factor and vascular endothelial growth factor are activated in the ischemic region of the myocardium, but not in normal regions [22,23]. Increased
expression of myocardial bFGF during ischemia has been shown in vivo by some [24], but not by other investigators [22]. Thus, although it is tempting to assume that the source for the detectable levels of bFGF found in our study population was the result of increased production and / or secretion from the ischemic myocardium, firm evidence to support this assumption is still lacking. In the course of angiogenesis, the vascular basement membrane is disrupted, resulting in the release of bFGF [2]. It could therefore be suggested that bFGF are detectable in patients in whom coronary collaterals are developing. In our study, detectable serum bFGF levels were not indicative of the presence of collateral circulation. We also found that serum bFGF levels were particularly detectable in patients with minimal coronary artery stenosis and in patients with less extensive coronary artery disease. These findings do not contradict the possibility that serum bFGF levels are increased due to myocardial angiogenesis and coronary collateral development, because collateral circulation may develop in the absence of obstructive coronary artery disease [25]. Moreover, since coronary angiography was performed at one point of time, we cannot determine the dynamics of collateral circulation development, and thus might have obtained samples from patients in different stages of collateral circulation development. Nevertheless, these data suggest that detectable serum bFGF levels may reflect processes unrelated to myocardial angiogenesis and development of collateral circulation. Detectable serum levels of bFGF in patients with ischemic heart disease may reflect active atherogenesis. Growth factors, including bFGF, are presumed to orchestrate the chain of events culminating in the formation of the atherosclerotic plaque [14]. Fibroblast growth factors (acidic and basic) have been implicated in the pathogenesis and disruption of atherosclerotic plaques [15–19]. Indeed, increased expression of bFGF, and more so of acidic fibroblast growth factor, have been exhibited in human atheromatous tissue [15–18]. Chen et al. [15] found bFGF in human coronary arteries with proliferative lesions, but not in fibrous plaques. Moreover, it is possible to attenuate smooth muscle proliferation in these lesions by adding a neutralizing antibody to bFGF or bFGF antisense oligonucleotides [15]. Simi-
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larly, Liau et al. [16] reported decreased mRNA for bFGF (and increased mRNA for acidic fibroblast growth factor) in advanced atheromatous lesions in hypercholesterolemic pigs, and increased mRNA for bFGF (and decreased mRNA for acidic fibroblast growth factor) in normal-appearing regions adjacent to these lesions. Therefore, it seems that bFGF plays a role in the early stages of plaque formation characterized by intense smooth muscle proliferation, and not in advanced atherosclerosis (fibrous plaque).
4.3. Limitations Serum bFGF levels were determined only once in our patients. It is possible that serum bFGF levels fluctuate, and thus we might have underestimated the prevalence of elevated bFGF levels among ischemic patients. A rise in bFGF levels may reflect decreased clearance or uptake, and / or increased production or secretion. In our study, we could not determine which of these mechanisms was responsible for the elevation in serum bFGF levels. Three, serum bFGF levels might not reflect tissue levels of bFGF. The correlation between local and systemic levels of bFGF remains to be determined in future studies. Four, differences in concomitant drug therapy may have influenced serum bFGF levels. To our knowledge, there are little data regarding the effect of antiischemic therapy on circulating bFGF levels.
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