Author + information
- Received October 20, 2004
- Revision received January 19, 2005
- Accepted January 25, 2005
- Published online November 1, 2005.
- Stephan Zbinden, MD,
- Rainer Zbinden, MD,
- Pascal Meier, MD,
- Stephan Windecker, MD and
- Christian Seiler, MD, FACC, FESC⁎ ()
- ↵⁎Reprint requests and correspondence:
Dr. Christian Seiler, Professor and Co-Chairman of Cardiology, University Hospital, CH-3010 Bern, Switzerland.
Objectives This study was designed to investigate the safety and efficacy of a short-term subcutaneous-only granulocyte-macrophage colony-stimulating factor (GM-CSF) protocol for coronary collateral growth promotion.
Background The safety and efficacy of an exclusively systemic application of GM-CSF in patients with coronary artery disease (CAD) and collateral artery promotion has not been studied so far.
Methods In 14 men (age 61 ± 11 years) with chronic stable CAD, the effect of GM-CSF (molgramostim) on quantitatively assessed collateral flow was tested in a randomized, double-blind, placebo-controlled fashion. The study protocol consisted of an invasive collateral flow index (CFI) measurement in a stenotic as well as a normal coronary artery before and after a two-week period with subcutaneous GM-CSF (10 μg/kg; n = 7) or placebo (n = 7). Collateral flow index was determined by simultaneous measurement of mean aortic, distal coronary occlusive, and central venous pressure.
Results Collateral flow index in all vessels changed from 0.116 ± 0.05 to 0.159 ± 0.07 in the GM-CSF group (p = 0.028) and from 0.166 ± 0.06 to 0.166 ± 0.04 in the placebo group (p = NS). The treatment-induced difference in CFI was +0.042 ± 0.05 in the GM-CSF group and −0.001 ± 0.04 in the placebo group (p = 0.035). Among 11 determined cytokines, chemokines, and their monocytic receptor concentrations, the treatment-induced change in CFI was predicted by the respective change in tumor necrosis factor-alpha concentration. Two of seven patients in the GM-CSF group and none in the placebo group suffered an acute coronary syndrome during the treatment period.
Conclusions A subcutaneous-only, short-term protocol of GM-CSF is effective in promoting coronary collateral artery growth among patients with CAD. However, the drug’s safety regarding the occurrence of acute coronary syndrome is questionable.
A revascularization strategy alternative to bypass surgery or angioplasty is warranted, both to control symptoms and to alter the course of severe coronary artery disease (CAD), because the traditional approaches are not suitable in 1 out of 5 to 3 patients. The promotion of preformed collateral arteries to a myocardial territory jeopardized by a blocked artery is such an option. Experimentally, there have been many candidates with angiogenic or arteriogenic properties (1), but in only one controlled clinical investigation using granulocyte-macrophage colony-stimulating factor (GM-CSF) has the promotion of large, conductive collateral arteries (arteriogenesis) been documented to be attainable (2). Conversely, inducing the growth of capillary-like collateral vessels (angiogenesis) in patients with CAD has not influenced myocardial perfusion (3,4). With pro-angiogenic and -arteriogenic substances, the risk of advancing atherosclerosis while promoting vascular detours has been criticized (5). During arteriogenesis, monocytes seem to preferentially adhere to the endothelium of vessels subject to high-velocity blood flow owing to a pressure gradient between a collateral supplying artery and a blocked vascular territory (i.e., homing). In this context, it is probably not necessary to deliver growth factors for monocyte stimulation locally, as performed in our recent study (2), but they can be given systemically.
On the basis of these considerations, the goal of the present controlled study was to investigate the safety and efficacy of a short-term subcutaneous-only GM-CSF protocol for coronary collateral growth promotion. The following hypotheses were tested: GM-CSF is safe and it augments directly obtained coronary collateral flow index (CFI) (Fig. 1).
Fourteen patients (age 61 ± 11 years, all men) with chronic stable one- (n = 4) or two-vessel (n = 10) CAD eligible for percutaneous coronary intervention (PCI) were included in the study. Patients were prospectively selected on the basis of the following criteria: 1) no previous transmural infarction in the myocardial area assessed for coronary collaterals, 2) normal left ventricular ejection fraction, 3) no congestive heart failure, 4) no baseline electrocardiogram (ECG) ST-segment abnormalities, 5) no signs of inflammatory illness, 6) absence of overt neoplastic disease, and 7) no diabetic retinopathy. Patients were randomly assigned to a two-week, double-blind protocol of subcutaneous GM-CSF (molgramostim; Novartis, Basel, Switzerland; n = 7) or placebo (n = 7). Collaterals were assessed invasively during balloon occlusion in a stenotic and a normal coronary artery (1 to 2 atm balloon inflation) at baseline before and immediately after the treatment period (i.e., before PCI). The study was terminated early because of two serious adverse cardiac events in the treatment arm of the study.
This investigation was approved by the institutional ethics committee, and the patients gave written informed consent to participate in the study.
Cardiac catheterization and coronary angiography
Patients underwent left heart catheterization from the right femoral approach. Aortic pressure was measured using a 6-F PCI guiding catheter. Central venous pressure was obtained via the right femoral vein. Left ventricular end-diastolic pressure was determined before PCI. Biplane left ventriculography was performed followed by biplane coronary angiography. Coronary artery stenoses were determined quantitatively as percent diameter narrowing.
Coronary collateral assessment
Coronary collaterals were assessed dichotomously according to the presence or absence of ECG signs of myocardial ischemia at the end of a one-minute balloon occlusion of the vessel of interest. Myocardial ischemia was defined as ST-segment changes >0.1 mV (Fig. 1).
Primary end point of the study
Coronary collateral flow relative to normal antegrade flow through the non-occluded vessel (CFI) was determined using coronary pressure measurements. A 0.014-in pressure monitoring angioplasty guidewire (Pressure Wave, Endosonics, Mountain View, California) was set at zero, calibrated, advanced through the guiding catheter, and positioned in the distal part of the vessel of interest. Collateral flow index was determined by simultaneous measurement of mean aortic pressure (Pao, mm Hg), the distal coronary artery pressure during balloon occlusion (Poccl, mm Hg), and the central venous pressure (CVP, mm Hg) (Fig. 1). Collateral flow index was calculated as (Poccl− CVP) divided by (Pao− CVP). The accuracy of pressure in comparison to Doppler-derived CFI measurements and to ECG signs of myocardial ischemia during occlusion has been documented previously (6).
Determination of progenitor cells, cytokines, chemokines, and their receptors
For quantitation of the CD34 messenger ribonucleic acid, a real-time quantitative polymerase chain reaction assay based on a specific set of primers and probe (Assays-on-Demand, Gene Expression Products) supplied by Applied Biosystems (Rotkreuz, Switzerland) was used.
Cytokines and chemokines
Concentrations of GM-CSF, monocyte chemoattractant protein-1 (MCP-1), basic fibroblast growth factor, and vascular endothelial growth factor were determined as immunoreactivity using a quantitative sandwich enzyme immunoassay technique (Quantikine, R and D Systems, Minneapolis, Minnesota). Concentrations of tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 were determined by immunometric assays (Immulite, DPC, Los Angeles, California) according to the manufacturer’s guidelines. Fractalkine concentration was assessed using enzyme-linked immunosorbent assay with mouse anti-human Fractalkine capture antibody and biotinylated mouse anti-human Fractalkine detection antibodies (DuoSet; R and D Systems, Minneapolis, Minnesota).
Cytokine and chemokine receptors
Cytokine and chemokine monocyte receptor concentrations (TNF-alpha receptor, TNFR1; MCP-1 receptor, CCR2; Fractalkine receptor, CXCR1) were determined by fluorescent-activated cell sorting analysis on CD14+ mononuclear cells.
Between-group comparisons of continuous clinical, hemodynamic, angiographic, blood analysis and collateral flow data were performed by a Mann-Whitney test. A chi-square test was used for comparison of categorical variables among the two study groups. Intraindividual comparison of baseline versus follow-up data was performed using Wilcoxon signed-rank test. Linear regression analysis was performed to assess an association between treatment-induced alterations of CD34+ cells, cytokines, chemokines, their receptors, and CFI changes. Parameters significantly related to CFI changes in this univariate regression analysis were entered in a multivariate stepwise regression analysis model for the determination of factors independently predicting CFI. Mean values ± SD are given.
Patient characteristics and clinical data at baseline
There were no statistically significant differences between the two groups regarding age of the patients, gender, body mass index, heart rate, or severity and duration of angina pectoris. There were no statistical differences in the frequency of cardiovascular risk factors and the use of acetylsalicylic acid, vasoactive drugs, statins, or diuretics (Table 1).
Hemodynamic, coronary structure, function, and collateral data at baseline
Mean blood pressure during vessel occlusion, left ventricular ejection fraction, end-diastolic pressure, central venous pressure, pressure during coronary occlusion in the stenotic and the normal vessel were similar between the study groups at baseline (Table 2).There were no differences among the groups in the number of vessels with relevant stenotic lesions, in the total number of hemodynamically relevant stenoses, in the vessel selected for PCI, in the angiographic severity of the treated stenosis, and in the hemodynamic degree of obstruction (fractional flow reserve) of the stenotic or normal vessel.
Qualitative and quantitative variables for the assessment of the collateral circulation were similar among the groups (Table 2).
Two patients in the GM-CSF group and none in the placebo group suffered an acute coronary syndrome with proximal occlusion of the left anterior descending coronary artery (at day 12 of the treatment protocol) and proximal occlusion of the right coronary artery (at day 9 of the treatment protocol) (Fig. 2).The vessel could be successfully recanalized in both individuals. Maximum creatine kinase levels following the event were 64 and 622 U/l. Patients of the GM-CSF group complained about any side effect in 6 of 7 instances and those in the placebo group did so in 2 of 7 cases (p < 0.05). Low fever temperatures occurred in 3 of 7 patients in the GM-CSF group and in none in the placebo group (p = NS). Skin rashes during treatment appeared in 7 of 7 cases in the GM-CSF group and in 2 of 7 placebo cases (p = 0.01).
Treatment-induced laboratory parameter and collateral flow changes
Total leucocyte count, neutrophils, eosinophils, and monocytes increased significantly in the GM-CSF group, whereas they remained statistically unchanged in the placebo group (Table 3).CD34+ cells showed a trend to decrease in the treatment group and to increase in the placebo group. Total and high-density lipoprotein cholesterol decreased significantly in the GM-CSF group, whereas they remained stable in the placebo group. Among the growth factor, cytokine, and chemokine concentrations obtained, the following increased significantly during follow-up in the treatment but not in the placebo group: GM-CSF, TNF-alpha, and MCP-1 (Table 3). The concentrations of basic fibroblast growth factor, vascular endothelial growth factor, and fractalkine did not alter relevantly either in the treatment or the placebo group. TNFR1 and CXCR1 monocyte receptor concentration was significantly up- and down-regulated, respectively in the GM-CSF but not in the placebo group (Table 4).The CCR2 receptor concentration remained statistically unchanged. Electrocardiographic signs of myocardial ischemia disappeared more often in the GM-CSF versus the placebo group (Table 4). Collateral flow index of all vessels increased significantly in the GM-CSF group, and it remained unaltered in the placebo group (Table 4, Fig. 3).A similar pattern was observed in the vascular subgroups of stenotic and normal coronary arteries. Among 11 variables (initial CFI, treatment-induced change [Δ] of neutrophils, eosinophils, monocytes, cholesterol, high-density lipoprotein, GM-CSF, TNF-alpha, MCP-1, TNFR1, CXCR1) examined by univariate linear regression analysis for their association to CFI change, ΔTNF-alpha was the only factor independently predictive of ΔCFI (Fig. 4).Tumor necrosis factor-alpha change was, itself, related to TNFR1 change: ΔTNF-alpha = 0.053 + 6.447ΔTNFR1; r2= 0.61, p < 0.0001.
The present study shows that a purely systemic application of GM-CSF is effective in promoting coronary collateral flow, and that this is related to enhanced TNF-alpha production with monocytic TNF receptor-1 up-regulation. The safety of the drug with regard to atherosclerotic plaque rupture in this study is questionable.
Mechanism of GM-CSF’s efficacy in arteriogenesis
The therapeutic use of a substance for the promotion of monocytes such as GM-CSF has been evaluated in one experimental and one clinical study (2,7). Buschmann et al. (7) found that a continuous infusion of GM-CSF into the stump of the acutely occluded femoral artery of rabbits enhanced the maximal blood flow of the hindquarter five-fold. The mechanism of action in that study was found to be the prolonged survival of monocytes. In the present study, the only independent factor influencing CFI change following GM-CSF was TNF-alpha along with an up-regulation of the corresponding receptor. This is in keeping with the results of earlier experimental studies on the role of this cytokine in arteriogenesis (8). It has also been hypothesized that GM-CSF could unfold its effect on arteriogenesis by releasing pluripotent stem cells from the bone marrow into the circulation, which would then be incorporated as endothelial cells into the growing collateral artery (7). Determination of CD34+ progenitor cells in the present study does not support this hypothesis, because GM-CSF did not relevantly alter the count of these cells. Nor does it undermine the possibility that progenitor cells play a role in arteriogenesis, because the time when the blood was collected for assessing CD34+ cells may have been inadequate for detecting their change. A specific group of progenitor cells, i.e., peripheral blood endothelial progenitor cells, has been found not to proliferate but to release pro-angiogenic growth factors and to be mainly derived from monocytes/macrophages (9). Similarly, a recent study has documented that bone-marrow-derived cells do not incorporate into the growing vasculature of mice (10). This indicates that there may be no need taking the “detour” of bone marrow mobilization or even transplantation to arrive at enhanced collateral vessels.
The role of GM-CSF in clinical arteriogenesis
Directly promoting peripheral or also bone marrow-derived monocytes by, for example, GM-CSF is a scientifically sound concept of collateral artery promotion acting via the production of an entire “cocktail” of cytokines. The few controlled clinical trials that have employed solitary angiogenic growth factors have failed to extrapolate the promising results from animal studies into the therapeutic area of human patients (3,4). This failure may not only be related to the selection of growth factors that induce capillary sprouting, but also to the choice of study end points (SPECT during vessel patency, exercise time) that are inadequate for selective assessment of collaterals (i.e., the parameter of interest hypothesized to be beneficially influenced by the substance under investigation).
Aside from a simplification of the subcutaneous-only study protocol, to find GM-CSF effective supports the hypothesis of “homing” of monocytes, i.e., the concept that they attach locally to vascular endothelium subject to augmented shear forces because of a pressure drop along a preformed collateral vessel. Alternatively, monocytes may “home” at vascular sites exposed to high shear forces for other reasons than a perfusion pressure gradient between collateral supplying and receiving artery, for example, at an atherosclerotic plaque obstructing the epicardial coronary artery.
GM-CSF and atherogenesis/atherogenic plaque rupture
Considering in this context the role of monocytes in atherogenesis, it is conceivable that the use of GM-CSF may translate into the rupture of atherosclerotic plaques alongside the growth of collateral arteries. Theoretically, a pro-atherogenic action of GM-CSF could be imagined via its MCP-1 elevating effect, which could be observed in the present study. Van Royen et al. (5) demonstrated in apolipoprotein E-deficient mice that local MCP-1 therapy in the ligated femoral artery augmented collateral artery formation and atherosclerotic plaque progression. In the present study, two patients in the GM-CSF group, but none in the placebo group, had had an acute coronary syndrome, very likely because of an atherosclerotic plaque rupture. Considering that “no-option” patients with extensive CAD are the most likely candidates for a therapy with GM-CSF, safety of the drug in relation to acute coronary syndromes cannot be guaranteed. This interpretation of the study results can also be extended to patients with subclinical CAD receiving GM-CSF for other indications.
The authors thank Caroline Zwicky, MD, and Elisabeth Oppliger, PhD, Hematology, University Hospital, Bern, Switzerland, for their support of the analysis of CD34 cells.
Supported by a grant from the Swiss National Science Foundation, #3200BO-100065/1. The first two authors contributed equally to this work.
- Abbreviations and Acronyms
- coronary artery disease
- collateral flow index
- central venous pressure
- granulocyte-macrophage colony-stimulating factor
- mean aortic pressure
- mean coronary artery occlusive pressure
- percutaneous coronary intervention
- tumor necrosis factor
- Received October 20, 2004.
- Revision received January 19, 2005.
- Accepted January 25, 2005.
- American College of Cardiology Foundation
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