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J Thorac Cardiovasc Surg 2006;132:891-899
© 2006 The American Association for Thoracic Surgery
Cardiopulmonary Support and Physiology |
a Department of Cardiovascular Surgery, Kyoto University, Graduate School of Medicine, Kyoto, Japan
b Human Brain Research Center, Kyoto University, Graduate School of Medicine, Kyoto, Japan
c Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan.
Received for publication December 22, 2005; revisions received June 5, 2006; accepted for publication June 22, 2006. * Address for reprints: Masashi Komeda, MD, PhD, Professor and Chairman, Department of Cardiovascular Surgery, Graduate School of Medicine, Kyoto University, 54 Shogoin Kawahara-machi, Sakyo-ku, Kyoto 606-8507, Japan. (Email: komelab{at}kuhp.kyoto-u.ac.jp).
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Abstract |
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Methods: Chronic ischemia was created in rabbits by placing a constrictor on the left circumflex artery. Four weeks later the animals were divided into 3 groups: group FG, in which a gelatin hydrogel sheet incorporating 100 µg of basic fibroblast growth factor was placed over the left circumflex region followed by covering with the omental flap including the intact gastroepiploic artery; group F, in which only the basic fibroblast growth factor sheet was placed; and group N, in which no treatment was done.
Results: Cine magnetic resonance imaging analysis showed a greater percentage wall thickening in the left circumflex region in group FG than in other groups (group FG, 49.2% ± 4.5%; group F, 41.2% ± 3.8%; group N, 32.1% ± 2.5%, P =.035, group FG vs group F). A colored microsphere assay showed higher perfusion in the left circumflex region in group FG than in group F. Perfusion in the left circumflex region was decreased after clamping the gastroepiploic artery pedicle in group FG (before clamping, 2.83 ± 0.72 mL · min–1 · g–1; after clamping, 1.93 ± 0.59 mL · min–1 · g–1; P < .01). In vivo angiography via gastroepiploic artery showed direct "to-and-fro" visible collaterals between the gastroepiploic and occluded left circumflex coronary arteries in group FG.
Conclusion: The combined growth factor therapy with an omental flap induced arteriogenesis and provided additional perfusion via the gastroepiploic artery to ameliorate regional dysfunction in the chronically ischemic myocardium.
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Introduction |
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As progress has been made in the basic studies on growth factors in the normal angiogenic process, the concept of therapeutic angiogenesis was developed as an alternative treatment for these patients over the past 2 decades.2 Preclinical animal studies with various growth factor delivery strategies have shown promising data.3,4 However, recent randomized double-blind clinical trials showed disappointing results with respect to therapeutic efficacy.5,6
Historically, before the advancement of cardiopulmonary bypass, the concept of employing an omental flap to provide revascularization for the ischemic myocardium was attempted in patients with ischemic heart disease. However, the therapeutic efficacy of omentopexy was not so efficient for rapid recovery. There exist the clinical limitations with the therapeutic efficacy of ometopexy as well as with that of growth factor therapy.7
To offer a more effective therapeutic option for these patients, we developed a combined method involving an omental flap and growth factor as a new alternative surgical method. We called this strategy "biologic coronary artery bypass grafting." Our previous study demonstrated that the method augmented the therapeutic efficacy of growth factor therapy using sustained-release basic fibroblast growth factor (bFGF) by applying a pedicled omental flap in a rabbit model of acute myocardial infarction, whereas omentopexy alone was not so effective as growth factor therapy.8 Regarding the clinical application, the purpose of the present study was to evaluate its therapeutic efficacy and to verify its superior therapeutic effect over growth factor therapy in the chronically ischemic myocardium.
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Materials and Methods |
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Each animal received two consecutive operations during the study. In the first operation, we created chronic myocardial ischemia by placing an ameroid constrictor. Four weeks after the first operation, animals were assigned into 3 groups as follows: group N (n = 8) received no additional treatment after creation of the myocardial ischemia; in group F (n = 8) a gelatin hydrogel sheet incorporating 100 µg of bFGF was placed over the epicardium of the ischemic area; and in group FG (n = 8) a gelatin hydrogel sheet incorporating 100 µg of bFGF was placed over the ischemic area, followed by covering with an omental flap. Four weeks after the second operation, animals were put to death with an overdose of pentobarbital to harvest the cardiac tissue for further assessments.
Anesthetic protocol for the surgical procedures and functional measurements
All surgical procedures in this study were performed with the animals under general anesthesia as described below. The rabbits were sedated with an intravenous injection of sodium pentobarbital (30 mg/kg) and then intubated with an end-tracheal tube (4.0-mm inner diameter, neonatal end-tracheal tube; Mallinckrodt Medical, St Louis, Mo) for mechanical ventilation (tidal volume of 10-15 mL and a minute ventilation rate of 40-60 breaths/min) (7025 Rodent Ventilator; Ugo Basile, Rome, Italy). General anesthesia was maintained with 1.0% to 2.0% of isoflurane mixed with room air. Rectal temperature was maintained at 38°C-39°C with a heat pad during the surgical procedures.
Creation of Chronic Myocardial Ischemia With an Ameroid Constrictor: The First Operation
We used a rabbit model of chronic myocardial ischemia according to the method previously described by Operschall and coworkers.9 In brief, a left thoracotomy was performed at the fourth intercostal space in a sterile manner. After the pericardium was opened, the main branch of the left circumflex (LCx) coronary artery was identified. We applied a commercially available hygroscopic ameroid constrictor specially designed for constricting rabbit coronary arteries (5-mm diameter, 1.5-mm height: Research Instruments SW, San Diego, Calif). We fixed it on the epicardial surface of the heart, surrounding the targeted coronary artery, with a 6-0 polypropylene suture placed around its circumference. The knot was softly tied to place the ameroid constrictor on the epicardial surface of the targeted artery preventing complete occlusion of the flow, as demonstrated by both electrocardiographic changes and visual blanching of the myocardium. Careful inspection was maintained for 10 minutes after placement of the constrictor. The thoracotomy was closed in layers, and residual air in the thoracic cavity was evacuated.
Echocardiographic measurements of time-course cardiac function
Left ventricular (LV) function was assessed by transthoracic echocardiography at each procedure (every 4 weeks) under general anesthesia as described above. A commercially available echocardiograph with a 7.5-MHz pediatric transducer (Vivid 7; GE Medical, Tokyo, Japan) was used for all studies to obtain serial images through a left parasternal approach. LV end-diastolic and end-systolic dimensions (LVEDD and LVESD, respectively) were measured with M-mode tracings from the short-axis view at the papillary muscle level. Fractional shorting (FS) was calculated from these data as follows: FS (%) = (LVEDD – LVESD)/(LVEDD) x 100. The data were averaged over 3 consecutive cardiac cycles. Another observer who was blinded to the treatment groups performed all measurements.
Preparation of the gelatin hydrogel sheet incorporating bFGF
Gelatin hydrogel sheets (Nitta Gelatin Co, Osaka, Japan) were prepared as described previously.10 In brief, the sheets were freeze-dried and trimmed in 5 x 5–mm squares and 0.7-mm thick, then impregnated with an aqueous solution containing 100 µg of human recombinant bFGF (Kaken Pharmaceutical Co, Tokyo, Japan). All the processes were conducted under sterile conditions.
Treatment: The Second Operation
Four weeks after the first operation, each animal in groups FG and F received 2 different types of treatment as the second operation. We excluded rabbits if they showed an FS more than 30% or less than 15% in the echocardiograhic assessment before the second operation.
In group F, a median sternotomy was performed to place the bFGF-incorporated hydrogel sheet (bFGF sheet) on the epicardium of the ischemic area (LCx region) by stitching around the edge of the sheet. In group FG, a small upper midline laparotomy and median sternotomy were performed to take the omentum out from the peritoneal space into the mediastinal space, preserving the arch structure of the left gastroepiploic artery (GEA). We created the hole at the diaphragm and passed the omental flap through the hole into the pericardial cavity. The bFGF sheet was placed on the epicardium of the ischemic area, followed by covering the sheet with the harvested omental flap.
Magnetic Resonance Imaging Analysis of Cardiac Performance
Four weeks after the second operation, we performed electrocardiographically gated cine magnetic resonance imaging (MRI) scans (Siemens Sonata 1.5 Tesla; Siemens Medical System, Erlangen, Germany) while the animals were anesthetized with an intravenous injection of sodium pentobarbital (30 mg/kg). A circular polarized extremity coil was wrapped around the chest for signal acquisition. Six sequential images of LV short-axis views every 2.5 mm were obtained to cover the entire LV volume from the base to the apex over 10 heartbeats. Custom designed software (Image J 1.3 version; Scion Corporation, Frederick, Md) was used to define myocardial borders and measure the wall thickness in the LCx region by the modified centerline method.11 The circumferential lengths at end-diastole and end-systole at the papillary muscle level were measured. LV volumes at end-systolic and end-diastolic phase (LVESV and LVEDV, respectively) were computed by the area-length method and used to calculate the LV ejection fraction (LVEF) as follows: LVEF (%) = (LVEDV – LVESV)/LVEDV x 100.12
Measurement of Regional Myocardial Blood Flow
Four weeks after the second operation, we evaluated regional myocardial blood flow in the ischemic area (LCx region) with a colored microsphere technique (DYE-TRCK; Triton Technology, Toronto, Ontario, Canada). A repeated left thoracotomy was performed to expose the left atrial appendage. After systemic heparinization (1000 IU heparin), 1.2 million red colored microspheres (15 µm in diameter) were injected into the left atrium for more than 30 seconds while reference blood samples were drawn from the descending aorta at a rate of 1.0 mL/min for 90 seconds. In group FG, 1.2 million yellow colored microspheres were consecutively injected to evaluate the GEA flow into the ischemic area in the same manner after clamping the GEA pedicle at the level of the diaphragm through a small laparotomy. After euthanasia with an overdose of pentobarbital, the LV was isolated and 3 short-axis transverse slices were cut out, while the transverse slices at the midventricular level were cut into 4 radial segments. According to the manufacturer's protocol, we extracted microspheres by potassium hydroxide digestion from the lateral midventricular segments and blood samples. The dyes were extracted from the spheres with 100 µL of dimethylformamide, and their concentrations were determined by spectrophotometry (UV-mini 1240; Shimazu. Co, Kyoto, Japan). The myocardial blood flow (Qs) was calculated as follows: Qs (mL · min–1 · g–1) = (As/Ar) Qr (mL/min)/Wt (g), where Qr represents with the withdrawal rate of the reference blood, As and Ar represent the absorbance in the sample tissue and reference blood, and Wt represents the tissue weight.
Histologic Analysis
Four weeks after the second operation, another series of 6 rabbits in each group was used for histologic analysis. After euthanasia, the heart was perfused at 100 mm Hg for 10 minutes with 10% formalin and then immersion-fixed in 4% formalin. We isolated the LV and cut out the short-axis transverse slice corresponding to the ischemic region situated 2 mm below the implanted ameroid constrictor. This transverse slice of LV at the papillary muscle level was embedded in paraffin and sectioned at 4-µm thickness. The primary mouse monoclonal antibody against 
–smooth muscle actin (clone 1A4; Sigma Chemical Co, St Louis, Mo) was incubated with the tissue section, followed by incubation with a biotin-rabbit anti-mouse immunoglobulin G. Tetramethylrhodamine isothiocyanate–conjugated secondary antibody was used to detect expression of –smooth muscle actin. The tissue sections were counterstained with hematoxylin and eosin. The numbers of arterioles were counted under a microscopic field (x100) to determine the arteriolar density. Five high-power fields were randomly selected for the vessel counts at the center of the lateral myocardial territories in each section. An arteriole was defined as a vessel in diameter more than 50 µm. Quantification was performed in a blinded manner with a minimum of 3 sections for each animal.
Microvascular Corrosion Cast
We made a microvascular corrosion cast to evaluate 3-dimensional collateral development in group FG, as previously described.13 Four weeks after the second operation, the resin (Mercox CL; Dainippon Ink Chemical, Tokyo, Japan) was injected antegradely into the celiac arterial trunk and retrogradely into the descending thoracic aorta after systemic heparinization (1000 IU heparin). The whole body was immersed in hot water for a few hours to solidify the resin in the blood vessels. The heart covered with the omental flap was carefully harvested and then placed into 10% sodium chlorate solution to corrode the residual adjacent tissue except for the arteries. This specimen was fixed with liquid nitrogen. After being coated with platinum-palladium, an image of collateral vessels was obtained by scanning electron microscopy (S4000; Hitachi Co, Tokyo, Japan).
In Vivo Angiography
A different series of 5 rabbits in group FG were anesthetized as described above. The right common carotid artery was cannulated with a 4F sheath introducer system (Goodtech; Goodman Co, Nagoya, Japan) after systemic heparinization (1000 IU heparin). A 3.2F catheter (Selecon PA catheter; Clinical Supply Co, Gifu, Japan) was selectively inserted into the celiac arterial trunk under fluoroscopy (OEC9800; GE Medical, Tokyo, Japan). The serial images of the collateral arteries were recorded at the rate of 30 frames per second with manual injection of 10 to 30 mL of diluted nonionic contrast medium (Iopamiron 300; Schering Co, Munich, Germany). Collateralization and myocardial blush were assessed qualitatively.
Statistical Analysis
All the data are shown as mean ± standard deviation. Statistical analyses were performed with the Stat-View software (SAS Institute Inc, Cary, NC). Comparisons of echocardiographic data among the groups were performed by 2-way repeated measures analysis of variance. Comparisons of other data among the groups were performed by 1-way analysis of variance. If significance was found for a group, a time effect, or a group-by-time interaction, differences between groups were specified with the Tukey-Kramer test for post hoc comparisons.
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Results |
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Time-course Changes of Global LV Function Assessed by Serial Transthoracic Echocardiography
Four weeks after the constrictor implantation, FS was significantly reduced in all groups. Four weeks after each treatment, group FG showed a greater FS increase than group F (P = .038 vs group F) (Figure 1, A). Four weeks after the constrictor implantation, LVEDD was increased in all groups. Four weeks after each treatment, group FG and group F showed a greater recovery in LVEDD than group N, but there was no significant difference between group FG and F (P = .143 vs group F, P = .008 vs group N) (Figure 1, B).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDiscussionReferences |
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The microsphere assay in the present study showed that the combined method provided more perfusion in the ischemic region than single growth factor therapy. Moreover, in the group treated by the combined method, we observed 32% reduction of the perfusion in the ischemic region after clamping the GEA. These results clearly demonstrated that the combined method provided additional perfusion to the ischemic region via the GEA. In the cine MRI analysis, the combined method showed a marked improvement in regional contractility in the ischemic region as expressed in percentage wall thickening, which is referred to as an index of mechanical function across all layers of the myocardium. Generally, providing adequate perfusion to all myocardial layers in the ischemic region is needed to improve regional dysfunction caused by ischemia.14 These findings demonstrated that the combined method can provide additional blood flow through the GEA to perfuse all myocardial layers of the region of chronic ischemia.
Perfusion reflects the result of successful collateral formation. The development of effective collateral vessels involves arteriogenesis, which is referred to as a process based on growth and remodeling into functional vessels.15 Arteriogenesis is considered to be more important than angiogenesis due to higher perfusion capacity.16 As the morphologic parameter for arteriogenesis, the immunohistologic analysis in the present study showed that the combined method developed more arterioles in the ischemic region compared with single growth factor therapy. In addition, the combined method developed sizable collateral vessels directly from the GEA, as shown in the vascular corrosion cast study. These results suggest that the combined method induced arteriogenesis to develop the collateral vessels directly from the GEA in the chronically ischemic myocardium.
As described in our previous study, our strategy for developing effective collaterals for advanced coronary lesions is to apply an omental flap involving the GEA as nondiseased donor artery with high perfusion capacity.8 The process of arteriogenesis is mediated via shear stress.15 The intracoronary collaterals induced by single growth factor therapy provide the inadequate limited flow through the obstructed native donor coronary arteries to induce arteriogenesis for developing effective collateral vessels.17 In contrast, an omental flap can provide the adequate additional flow via the GEA as an unobstructed donor artery. From these considerations, the GEA involved in an omental flap can play an important role as an independent, unobstructed extracardiac blood source to provide additional collateral flow (shear stress) for enhancing arteriogenic activity in the chronically ischemic region.
Conversely, from the results of our studies, it is reasonable to conjecture that the combined method may enhance the angiogenic efficacy of omentopexy by interposing the bFGF sheet. As demonstrated by our previous study, the therapeutic efficacy of omentopexy alone was inferior to that of single growth factor therapy with bFGF sheet.8 Five decades ago, omentopexy was used to create collaterals from the GEA. However, in some cases, it took several years to develop effective ones.18 These facts suggest that the clinical disadvantage of omentopexy is its therapeutic inefficiency. The interposed bFGF sheet possibly enhances the angiogenic effects of the superimposed omental flap in the combined method. It can accelerate the formation of collaterals between native coronary arteries and omental tissue to achieve the vessel connections directly from the GEA.
Omental flap itself has been an attractive tissue for cardiothoracic surgeons to stimulate revascularization of ischemic tissue.19 Recent basic studies have elucidated the mechanism of the angiogenic action induced by omental tissue. The adipocytes in the omental tissue release a number of angiogenic growth factors, such as vascular endothelial growth factor and bFGF. This fact suggests that an omental flap can act as a physiologic exogenous source of multiangiogenic factors that are synergistically involved in the process of arteriogenesis.20,21 Moreover, the interposed bFGF sheet possibly can augment the expression of other growth factors in the omental tissue.22 However, further investigation is needed to clarify the participation of the additive growth factors released from the omental tissue in the process of collateral formation from the GEA in the combined method.
Other experimental studies were reported to enhance the effects of omentopexy. Ruel and associates23 demonstrated the excellent angiogenic effect of a gastric submucosal patch as an endogenous source of growth factors in a swine model of chronic myocardial ischemia. Kanamori and colleagues24 showed that omentopexy enhanced the angiogenic effect of cell therapy in a swine model of acute myocardial infarction. Compared with these studies, the combined method in the present study is safer and less invasive because it does not need a gastrectomy or bone marrow aspiration.
Several limitations of this study must be addressed. First, we did not quantitatively evaluate the functional capacity of developed collaterals in this study. However, the identification of angiographically visible "to-and-fro" collaterals can be considered as the qualitative evidence of functional collateral formation. Second, there existed a variation in the myocardial ischemia in the animal model of chronic ischemia with an Ameroid constrictor, although we designed the study to minimize this variation. As a further study, the efficacy of the method must be evaluated in hyperlipidemic or diabetic animal models because endothelial dysfunction resulting from these pathologic factors is one of the most important obstacles to achieving clinical therapeutic angiogenesis.25,26
In conclusion, we demonstrated that the combined method developed sizable collateral vessels directly from the GEA to ameliorate regional myocardial dysfunction in the chronically ischemic myocardium. We verified that the effect of the present method is superior to that of single angiogenic protein therapy. The present method may improve the clinical limitations in therapeutic angiogenesis and expand the surgical indication for patients with severe coronary artery disease.
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Discussion |
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I have 3 questions for you.
It is widely known that Vineberg-type procedures, be with it the internal thoracic artery, as has been described for several decades, or as now described, will increase myocardial blood flow. Do you have any data with the GEA control, that is, without using bFGF, to describe the relative contribution of the GEA alone as opposed to your bFGF angiogenic or arteriogenic therapies?
Second, we have previously demonstrated that the omentum is an extremely rich supply of vascular endothelial growth factor; in fact, it is the highest concentrations in the body. This presumably accounts for its role in abdominal healing, for example, and the use of omental flaps in general. Do you have any data looking at vascular endothelial growth factor alone as opposed to the omentum as your angiogenic/arteriogenic supplement?
Finally, similar work in this regard has been performed by Cohn and his associates, and has been reported previously, using essentially a very similar model, GEA as a Vineberg-type procedure. Do you have any information or are you aware of Dr Cohn's work compared with your own? Potentially that would provide some insights into the relative contribution of the GEA.
Dr Takaba. To answer your first question, we have investigated just omentopexy alone in a previous study of an acute myocardial infarction model. However, the effect of just omentopexy was lower than angiogenic factor alone. So now we can demonstrate just omentopexy.
Concerning your second question, basically we investigated bFGF, and bFGF is investigated for the effect of this. We have data for this.
Please repeat the third question.
Dr Rosengart. Are you familiar with Dr Cohn's prior work with a similar model in this area?
Dr Sellke. He used a gastric patch, based on the GEA, and did the same thing without the growth factor, but he found that there was increased perfusion in the chronically ischemic territory. Are you familiar with that?
Dr Takaba. I am not. Sorry.
Supplementary data
Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jtcvs.2006.06.023.
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Acknowledgments |
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Footnotes |
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Read at the Eighty-fifth Annual Meeting of The American Association for Thoracic Surgery, San Francisco, Calif, April 10-13, 2005.
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  M. Komeda, A. Marui, K. Tambara, M. Yamamoto, Y. Saji, T. Nishina, and Y. Tabata Biologic anastomosis: The first case of biologic coronary bypass surgery J. Thorac. Cardiovasc. Surg., September 1, 2009; 138(3): 775 - 777. [Full Text] [PDF]
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