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Ann Thorac Surg 2004;77:812-818
© 2004 The Society of Thoracic Surgeons
a Departments of Surgery and Medicine, Divisions of Cardiovascular and Thoracic Surgery, Duke University Medical Center, Durham, NC, USA
b Department of Cardiology, Duke University Medical Center, Durham, NC, USA
c Department of Radiology, Duke University Medical Center, Durham, North Carolina, USA
Accepted for publication September 8, 2003.
* Address reprint requests to Dr Hughes, Box 31224, Duke University Medical Center, Durham, NC 27710, USA
e-mail: chadh{at}duke.edu
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Abstract |
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METHODS: Mini-swine with proximal 90% left circumflex (LCx) coronary stenosis subtending chronically ischemic, viable (hibernating) myocardium by positron emission tomography (PET) and dobutamine stress echocardiography (DSE) were randomized to IM bFGF (n = 5), IM VEGF165 (n = 5), IV VEGF165 (n = 5), IM vehicle (n = 5), or sham redo-thoracotomy (n = 4). The bFGF protein was administered in a total dose of 1.35 µg divided into 30 IM injections. IM VEGF165 protein was administered in a total dose of 15 µg/kg divided into 30 injections; IV VEGF165 was given at a dose of 50 ng · kg-1 · min-1 for 200 minutes at three 72-hour intervals (30 µg/kg total dose). After 3 and 6 months the PET and DSE studies were repeated, and the animals were sacrificed for tissue vascular density and angiogenic protein analysis.
RESULTS: Myocardial blood flow (MBF) by PET was significantly improved 3 months posttreatment in the IM bFGF and IM VEGF165 groups, differences that were sustained at 6 months. There was no significant increase in MBF 3-months posttreatment in the IV VEGF165 group; however, at 6 months MBF was significantly improved. No change in MBF was seen in the IM vehicle or sham groups. Regional wall motion at rest and peak stress in the LCx region demonstrated small but statistically significant improvements by 6 months in the IM bFGF and IV VEGF165 groups only; no improvement was seen in the IM VEGF165, IM vehicle, or sham groups. Quantitative vascular density was significantly increased in the LCx regions of all treatment groups (IM bFGF, IM VEGF165, IV VEGF165) 6-months postoperatively. No significant increase in LCx region myocardial bFGF or VEGF protein levels was seen in the treated animals at 6 months.
CONCLUSIONS: The IM bFGF, IM VEGF165, and IV VEGF165 all improve regional perfusion and vascular density 6-months posttherapy in the animal model utilized. Functional improvements were less consistent. Both bFGF and VEGF165 may be useful therapies for improving regional perfusion in chronically ischemic myocardium, although combination therapy with additional growth factors or cellular therapies may be necessary if concomitant improvements in function are to be seen.
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Material and methods |
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Partial coronary occlusion model
Using a previously described porcine model of chronic hibernating myocardium [17, 18], animals underwent creation of a high-grade stenosis of the proximal left circumflex (LCx) coronary artery. A hydraulic occluder and 2-mm ultrasonic flow probe (Transonic Systems, Inc., Ithaca, NY) were placed around the proximal LCx, and 3 days postoperatively the occluder was inflated to reduce resting blood flow in the LCx downstream from the occluder to 10% of baseline as assessed using the implanted flow probe. Animals were then kept in this low-flow state for the duration of the experiment as previously described [17]. Animals were medicated with aspirin (650 mg daily) throughout the entire experiment.
Positron emission tomography and dobutamine stress echocardiography
After 30 days in the low-flow state, animals underwent positron emission tomography (PET) and dobutamine stress echocardiography (DSE) to document the presence of hibernating myocardium in the LCx distribution. After an overnight fast, dynamic PET imaging of the heart using 13N-ammonia and 18F-fluorodeoxyglucose (FDG) was performed as previously described [17, 18] to obtain regional estimates of myocardial blood flow (MBF; mL/g per minute) and glucose utilization (nmol/g per minute). The PET scans were interpreted as demonstrating hibernating myocardium if reduced absolute values of MBF were noted in the lateral and posteroinferior walls of the left ventricle supplied by the LCx, accompanied by normal or increased FDG uptake in these same regions (when compared with the nonischemic septum) [17–19].
Dobutamine stress echocardiography was performed in 3-minutes stages with incremental doses of dobutamine beginning with 5 µg · kg-1 · min-1 and increasing to 40 µg · kg-1 · min-1 as previously described [17, 18]. Based on a standard 16-segment model [20], wall motion was graded as follows: 1 = normal; 2 = hypokinetic (reduced systolic wall thickening); 3 = akinetic (absent systolic wall thickening); or 4 = dyskinetic (outward systolic wall motion). Regional wall motion score index (WMSI) was calculated at rest, low dose, and peak stress. Echocardiograms were interpreted in a blinded manner by a cardiologist with expertise in stress echocardiography. Using DSE, viability in the LCx region was defined as an improvement in systolic wall thickening with low-dose dobutamine in myocardial regions with severe hypocontractility at rest. Viable segments were considered ischemic if systolic wall motion deteriorated with stress (biphasic response) [17, 18].
Growth factor delivery
Once hibernating myocardium in the LCx distribution was demonstrated by PET and DSE, animals were randomized to one of the following five groups: IM bFGF (n = 5); IM VEGF165 (n = 5); IV VEGF165 (n = 5); IM vehicle (n = 5); or sham redo-thoracotomy (n = 4). All procedures were performed within 3 days of completion of the baseline PET and DSE studies. Recombinant human bFGF was supplied by the Chiron Corporation (Emeryville, CA) and recombinant human VEGF165 by Genentech, Incorporated (South San Francisco, CA).
For the IM bFGF group, recombinant human bFGF was administered at a total dose of 1.35 µg to treat the entire LCx region using 30 100-µL intramyocardial injections at 1-cm intervals. Similarly in the IM VEGF165 animals, recombinant human VEGF165 was administered at a total dose of 15 µg/kg to treat the entire LCx region using 30 100-µL intramyocardial injections at 1-cm intervals. All injections were performed using a 27-gauge needle into the midmyocardial level. In the IV VEGF165 group, animals underwent an infusion of recombinant human VEGF165 given through a catheter in an ear vein at a dose of 50 ng · kg-1 · min-1 over 200 minutes. This dosing was repeated 72- and 144-hours later (total dose 30 µg/kg). The IM vehicle control group animals underwent 30 100-µL intramyocardial LCx territory injections of delivery vehicle as described above. Those animals randomized to the sham redo-thoracotomy control group underwent an identical repeat thoracotomy where the pericardium was opened, but no IM injection performed. In all groups, continuous LCx occlusion was confirmed postoperatively by weekly monitoring with the flow probe.
Follow-up PET and DSE
Three and 6 months after the IM bFGF, IM VEGF165, IV VEGF165, or IM vehicle the animals underwent repeat PET and DSE. Repeat studies were performed at 6 months only in the sham redo-thoracotomy group. To allow comparisons between studies performed at baseline and follow-up and to correct for the known interstudy variability of absolute values of MBF by PET [21], normalization of the data were performed using previously described techniques [22, 23]. For each PET study, sectors representing the anterior septum were used as the normal reference segments [17, 18]. 13N-ammonia activity in the sectors representing the LCx distribution were then expressed as a percentage of the activity measured in the reference segments.
Vascular density analysis
Animals were euthanized 6 months after IM bFGF, IM VEGF165, IV VEGF165, IM vehicle, or sham redo-thoracotomy for histochemical staining to assess overall vascular density in the LCx region. Angiogenesis was assessed using endogenous endothelial alkaline phosphatase staining on unfixed frozen sections as previously described [22–25]. Endogenous endothelial alkaline phosphatase staining intensity was measured using an image analysis system (Olympus IX70 inverted microscope, Melville, NY; Optronics DEI-750 image-capturing hardware, Goleta, CA; PowerTower Pro 180 CPU, Round Rock, TX). Images were captured using Adobe Premiere (Adobe Systems Inc., San Jose, CA) and quantified using NIH image software (NIH, Bethesda, MD). Four randomly selected transmural samples from the LCx region were analyzed per animal. Six random high-power (200x) fields were examined per sample and overall vascular density quantitated in a blinded fashion by two independent observers using previously described techniques [23–25].
Myocardial bFGF and VEGF tissue protein levels
At the time of sacrifice, 50-mg sections of myocardium were excised from the center of the LCx region, weighed, pulverized in liquid nitrogen, and homogenized in 3 mL of 10-mmol/L tris (hydroxymethyl) aminomethane (Tris; pH 7.4) and 100-mmol/L NaCl using a Brinkmann Polytron (Westbury, NY). The suspension was centrifuged twice at 8000g and 4°C for 15 min. Total protein concentration of the supernatant was determined by the Bradford assay (Bio-Rad Laboratories, Hercules, CA). Myocardial bFGF and VEGF165 protein levels were then determined by a standard enzyme-linked immunoabsorbent assay method (R&D Systems, Inc., Minneapolis, MN).
Statistical analysis
All data are presented as the mean ± standard error. MBF and glucose utilization by PET, as well as WMSI by DSE were compared within groups using a paired Student's t test with a Bonferroni correction for multiple comparisons. One-way between-groups analysis of variance (ANOVA) was used to compare MBF, WMSI, vascular density, and myocardial protein levels between groups. Statistical significance was considered a p value less than 0.05.
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Quantitative vascular density analysis (Fig 3) revealed a significant increase (p < 0.05) in vascular density in the bFGF (5.1 ± 0.1 arbitrary units), IV VEGF165 (2.6 ± 0.1 arbitrary units), and IM VEGF165 (2.1 ± 0.1 arbitrary units) groups compared with sham animals (1.0 ± 0.1 arbitrary units). No significant difference was seen in the IM vehicle group (1.7 ± 0.1 arbitrary units) versus the sham group.
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Another unique feature of the present study is the dose of bFGF used (total dose 1.35 µg) is much less than all prior clinical and experimental reports, which have used doses ranging from 4 µg to upward of 14 mg [3–8, 11, 16, 27]. Given the concerns over hypotension, hematologic toxicity, and renal injury associated with higher doses of bFGF [6, 27], this lower and apparently efficacious dose may provide a greater margin of safety, although we did not specifically examine end-organ toxicity in this study.
This experimental study is the first to suggest efficacy for IV administered VEGF protein. Although, as has been pointed out in several recent reviews [26, 28], intravenous delivery may be preferred for ease of use and applicability to large patient populations, systemic recirculation and lack of sustained tissue exposure to the angiogenic protein may limit the usefulness of this approach. Prior work using the same infusion protocol as in this study (0.05 µg · kg-1 · min-1 x 200 minutes) failed to demonstrate efficacy for the intravenous administration of VEGF165 protein [29]. However, unlike the present work, that study used only a single infusion of VEGF protein (total dose 10 µg/kg), whereas this study used a total of three infusions 72-hours apart (total dose 30 µg/kg). Presumably, the higher total dose, as well as repeated dosing, account for the different results observed.
In addition, the prior study [29] assessed efficacy at 3-weeks posttreatment whereas the present study had 3- and 6-month endpoints. Interestingly, no significant change in regional perfusion or function was seen at the 3-month timepoint in the present study, with improvement delayed until 6-months after IV VEGF165 protein therapy. Whether similar results would have been seen in the prior study (using only a single dose of IV VEGF165) had the temporal endpoint been longer remains unknown. Because, in general, less than 1% of intravenously administered angiogenic protein can be recovered from ischemic myocardium 1-hour after administration [30], repeated dosing and higher doses than utilized with direct intramyocardial injection (where up to 30% of the injectate is recovered at 1 hour) may be necessary to produce therapeutic tissue levels of growth factor. The continued improvement up to 6 months in the IV VEGF165 animals is consistent with the results of the VEGF in ischemia for vascular angiogenesis (VIVA) trial where improvement in patients' symptom status was seen up to 6-months after treatment [15].
Improvements in regional function following angiogenic protein therapy in the present study were less impressive than changes in perfusion. There was no improvement in rest function 3-months posttreatment in any group and only small improvements in the IM bFGF and IV VEGF165 animals at 6 months. Similarly, peak stress wall motion improved slightly, although statistically significant, only in the bFGF animals at 3 months. By 6 months, improvement in the IV VEGF165 animals was seen as well. Whether this relative lack of functional improvement represents a deficiency of angiogenic protein therapy or is specific to the model tested is unknown.
As has been pointed out in a recent review [1], chronically ischemic yet viable myocardium is characterized by progressive myocyte apoptosis, ventricular remodeling, and structural alterations including loss of contractile material within cardiomyocytes, and increases in the amount of interstitial connective tissue and studies have demonstrated that patients with more advanced cardiomyocyte deterioration have less return of function after revascularization than those with less advanced changes [1]. Consequently, given the progressive nature of these phenotypic alterations in the absence of revascularization, functional recovery in long-standing hibernating myocardium after proangiogenic therapy may be limited despite improvements in regional perfusion.
No increase in myocardial protein levels of either bFGF or VEGF was seen 6 months posttreatment despite significantly higher vascular density in the LCx distribution of the left ventricle in the treatment groups. Although, given the relatively small number of animals in each group, a potential type II error cannot be ruled out, these results are not entirely unexpected as any upregulation of bFGF or VEGF myocardial protein levels likely would have occurred earlier in the 6 month time course and not necessarily be sustained, especially given the limited tissue half-life of angiogenic proteins as noted above. In addition, there was not a clear correlation between observed vascular density and improvements in regional perfusion or function in the treatment groups, with greater neovascularization in the bFGF versus VEGF animals despite similar increases in blood flow and function. Further investigation is necessary to clearly elucidate the mechanism of angiogenesis following protein delivery.
Two large phase II prospective, randomized clinical trials investigating the use of bFGF (FGF initiating revascularization trial, or FIRST trial) [16] and VEGF165 (VIVA trial) [15] protein have been performed. Both have been considered negative trials because no overall benefit in nuclear perfusion or exercise time was seen, although there did appear to be some benefit with regard to anginal symptoms. The reason for the discrepancy between the results seen in clinical and experimental studies [3–14] is unknown but potentially may relate to differences between the animal models used and human patients [1]. The experimental models typically used in these studies involve young, healthy animals with single vessel coronary disease where the remaining vessels are normal and thus potentially more able to sprout collateral vessels capable of improving myocardial blood flow to the ischemic regions. These differences may explain the disparate results seen to date in animal versus human studies [1].
In summary, intramyocardially administered bFGF, VEGF165, and systemically administered VEGF165 protein in the dosing regimen used appear to improve regional perfusion and vascular density 6-months posttherapy; functional improvements were less consistent. Both bFGF and VEGF165 may be useful therapies for improving regional perfusion in chronically ischemic myocardium. However, further work is needed, especially given the relative lack of improvement in regional function as well as the less impressive results of clinical trials performed to date. As pointed out in a recent editorial [31], given the complex and highly ordered physiologic regulation involved in the process of angiogenesis, it is unlikely that any single growth factor will be the "magic bullet" to cure end-stage coronary disease. Rather, combination therapy with multiple growth factors, mechanical means such as transmyocardial laser revascularization [32], or cell transplantation [33] may be necessary before consistent clinical benefits are seen and these therapies become widely accepted.
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= baseline; 
= 3-month follow-up; 
= 6-month follow-up. (bFGF = basic fibroblast growth factor; IM = intramyocardial; IV = intravenous; LCx = left circumflex; MBF = myocardial blood flow; PET = positron emission tomography; VEGF = vascular endothelial growth factor.)
