| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Ann Thorac Surg 2007;83:1110-1119
© 2007 The Society of Thoracic Surgeons
Division of Cardiovascular Surgery, Toronto General Hospital, Department of Surgery, University of Toronto, and Heart and Stroke Foundation/Richard Lewar Centre of Excellence, Toronto, Ontario, Canada
Accepted for publication October 20, 2006.
* Address correspondence to Dr Yau, Division of Cardiovascular Surgery, Toronto General Hospital, University Health Network, 4N-470, 200 Elizabeth St, Toronto, ON M5G 2C4, Canada (Email: terry.yau{at}uhn.on.ca).
 |
Abstract |
|---|
 Top Abstract IntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Methods: Female Lewis rats underwent coronary ligation 3 weeks before transplantation with male donor BMC, BMC transfected with VEGF (BMC+VEGF), bFGF (BMC+bFGF), VEGF and bFGF (BMC+VEGF+bFGF), or medium (control) (n = 3 each group at 3 days, 1 week and 2 weeks; n = 6 each group at 4 weeks; n = 75 total). Three days, 1 week, 2 weeks, and 4 weeks after transplantation, transgene expression was quantitated by real-time polymerase chain reaction, angiogenesis by quantitative histology, and LV function by echocardiography. At 4 weeks, regional perfusion was quantitated with microspheres.
Results: The VEGF and bFGF were expressed transiently over 4 weeks. At 1 week, VEGF expression was greatest in BMC+VEGF and BMC+VEGF+bFGF hearts (p < 0.05), while bFGF expression was greatest in BMC+bFGF and BMC+VEGF+bFGF rats (p < 0.05). Regional perfusion and vascular densities in the scar were lowest in control, intermediate in BMC, BMC+VEGF, BMC+bFGF, and greatest in BMC+VEGF+bFGF (p < 0.05). Four weeks after transplantation, LV ejection fraction was lowest in control, intermediate in BMC, BMC+VEGF and BMC+bFGF, and greatest in BMC+VEGF+bFGF (p < 0.05).
Conclusions: The VEGF and bFGF transgenes were expressed transiently and exerted a powerful synergism on the angiogenic effect of cell transplantation, but did not normalize perfusion or function. The future of cell transplantation may lie in multimodal cell-based gene therapy, in combination with other novel therapies.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
In these experiments, our objective was to determine whether the known synergism of VEGF and basic fibroblast growth factor (bFGF) on angiogenesis [5–8] could be utilized to further enhance the angiogenic effect of cell-based gene therapy. Another objective was to determine whether the combination of cell transplantation with two angiogenic transgenes, delivered 3 weeks after myocardial infarction, could normalize left ventricular (LV) function and perfusion.
|
Material and Methods |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Bone Marrow Cell Isolation
Donor bone marrow cells (BMC) were isolated from the femurs of male rats and cultured in 100-mm dishes in Iscove’s modified Dulbecco’s medium with 10% fetal bovine serum for 7 to 10 days before harvesting (at 60% to 70% confluence) for transfection and transplantation. The cultures were partially depleted of erythroid progenitor cells by removal of nonadherent cells with each change of medium, every 3 days. No further fractionation of BMC was performed.
Cell Transfection
Cells were transfected ex vivo, by a lipid-based technique, with a plasmid encoding VEGF165 (pCEP4-VEGF) or bFGF (pcDNA3.1-bFGF), or both, 24 hours before transplantation [1–4]. Transfection efficiencies were monitored in a subset of plates by cotransfection with pEGFP-N2 (BD Biosciences Clontech, Palo Alto, California), encoding green fluorescence protein.
Cell Transplantation
Three weeks after infarct creation, rats were randomly divided into five experimental groups and injected with culture medium alone (control), or transplanted with 3 x 106 untransfected BMC, BMC transfected with VEGF (BMC+VEGF), BMC transfected with bFGF (BMC+bFGF), or BMC transfected with VEGF and bFGF (BMC+VEGF+ bFGF) into multiple points in the center of the infarct (n = 3 each group at 3 days, 1 week, and 2 weeks; n = 6 each group at 4 weeks; n = 75 total) [1–4]. Rats were sacrificed, and the hearts were excised 3 days, 1 week, 2 weeks, or 4 weeks after cell transplantation [1–3]. The atria and the right ventricular free wall were excised, and the LV was divided into the scar zone (transmural scar), the border zone (partial-thickness scar containing both fibrous tissue and surviving muscle) and the normal myocardium.
RNA Isolation and Reverse Transcription
Myocardial specimens were snap-frozen in liquid nitrogen and powdered. A portion of each specimen was used immediately for total RNA isolation, whereas the remainder was stored at –80°C for subsequent analysis of protein levels. Total RNA was isolated with TRIzol RNA extraction reagents (Invitrogen Corp, Carlsbad, California). Messenger RNA in this specimen was reverse transcribed to single-strand complementary DNA (cDNA) with SuperScript II reverse transcriptase (Invitrogen Corp) [2, 3].
Quantitation of VEGF and bFGF mRNA by Real-Time Polymerase Chain Reaction
Quantitation of VEGF and bFGF messenger RNA expression was carried out by means of real-time polymerase chain reaction (PCR) [2, 3]. Specific PCR primers were designed from VEGF and bFGF GeneBank sequences. VEGF primers (antisense, 5'-TCATGGTTGTCTATCAGCGCAG-3'; sense, 5'-GCACACAGGATGGCTTGAAGAT-3') generated a 107-base pair fragment. bFGF primers (antisense, 5'-CCCGTTTTGGATCCGAGTTTA-3'; sense, 5'-GAACGCCTGGAGTCCAATAACTAC-3') generated a 107-base pair fragment. Standard PCR was first performed with these primers using single-strand cDNA from the sample as a template. Gel electrophoresis confirmed that each PCR product comprised a single band of the correct size, which was excised and sequenced to confirm its identity. The purified products were quantitated spectrophotometrically for use as standards for subsequent real-time PCR assays, which were performed with these primers [2, 3].
Immunohistochemical Assays
The Sca-1, CD45, and CD34 were localized in cultured BMC by immunohistochemical double staining with a laser confocal microscopy system (Bio-Rad, Richmond, California), according to the manufacturer’s protocols (Molecular Probes, Eugene, Oregon) [2, 3]. Slides were incubated with phycoerythrin-labeled anti-Sca-1 antibodies (1:100), Alexa Fluor 488-labeled anti-CD45 antibodies (1:100), and Cy5-labeled anti-CD34 antibodies (1:100; all Molecular Probes) overnight at 4°C.
Quantitative Histology
Myocardial specimens were fixed in formalin, embedded in paraffin, and sectioned into 6-µm-thick slices. Samples were stained with hematoxylin and eosin for morphologic evaluation of the scar and transplanted cells (Sigma Diagnostics, St. Louis, Missouri) or immunohistochemical staining with antibodies against factor VIII, to facilitate quantitation of vascular density. The number of vessels per medium power field (x400) in the scar, border zone, or normal myocardium was counted by two masked observers in five fields per slide, and the mean number of vessels per field subjected to analysis.
Microsphere Analysis of Regional Blood Flow
Regional myocardial perfusion was evaluated by FluoSpheres polystyrene microspheres in 3 rats per group, 4 weeks after cell transplantation (n = 3 per group x 5 groups x 1 timepoint = 15). Under general anesthesia, the heart and great vessels were exposed through a median sternotomy. The ascending aorta was ligated and the right atrium was incised. One milliliter of a suspension containing 3.6 x 106 10 µm yellow-green fluorescent microspheres (Molecular Probes) were injected into the ascending aorta over 60 seconds. The heart was then excised, the atria and right ventricle removed, and the left ventricle was divided into scar, border zone, and normal regions. The weight of each specimen was recorded before digestion with 4 M potassium hydroxide (300 µL per 100 mg tissue) at room temperature for 24 hours. After complete digestion of the myocardial specimens, the microspheres were extracted and fluorescent intensities measured in a CytoFluor Multi-Well Plate Reader Series 4000 (PerSeptive Biosystems, Stafford, Texas).
Left Ventricular Function
Left ventricular function was evaluated in vivo in rats by transthoracic echocardiography (Siemens ACUSON Sequoia C256, Malvern, PA), as described by Sahn and colleagues [9] and Schiller and associates [10].
Statistical Analysis
Data are presented as mean ± SD. Categorical data were subjected to 
2 analyses, and continuous data to analysis of variance (ANOVA). When the ANOVA F value was significant, Duncan’s multiple range test was employed to specify differences.
|
Results |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
|
|
|
|
|
|
|
|
|
|
|
|
|
Comment |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
The enhanced angiogenesis induced by transplantation of VEGF-transfected cells was not, however, sufficient to normalize either perfusion or function [1]. Because angiogenesis is a complex process involving multiple cytokines, we hypothesized that augmentation of the angiogenic stimulus, through the well-described synergism of VEGF and bFGF [5–8], could further increase both regional perfusion and function. We utilized a model of myocardial infarction in syngeneic Lewis rats to eliminate the issue of rejection of the transplanted cells [2, 3]. Because previous experiments have demonstrated that transplanted BMC can improve postinfarction LV function [14], and that BMC may have greater plasticity in myocardial repair than heart cells [15], we utilized BMC as our transgene delivery vehicles.
As we had previously observed with a VEGF transgene alone, the expression of the VEGF and bFGF transgenes after transplantation of the BMC was limited spatially to the scar and border zone, and temporally to a 4-week period. Expression of VEGF in the BMC+VEGF group induced a moderate increase in bFGF expression in the border zone of those hearts, and similarly, a moderate increase in VEGF expression was noted in the border zone of the BMC+bFGF hearts. The VEGF and bFGF mRNA levels did not differ between any of the groups in the normal myocardium. We have previously noted that transplantation of unmodified heart cells or skeletal myoblasts results in a moderate but appreciable increase in VEGF expression compared with controls receiving only an injection of culture medium [2]. In contrast, transplantation of unmodified BMC in this study did not result in increased VEGF expression. There was a small and nonsignificant increase in bFGF expression in the scar and border zone of BMC rats.
Transgene expression of VEGF and bFGF peaked at 1 week, before the peak in vascular densities, which was observed 2 weeks after transplantation. Vascular densities were stable from 2 weeks to 4 weeks, the last timepoint in our study, when VEGF and bFGF expression had declined to baseline levels. In other studies, we have noted that angiogenesis in response to transiently VEGF-overexpressing heart cells and skeletal myoblasts persists for at least 6 months, and does not result in angioma formation [16]. However, our current data do not allow us to comment on whether BMC expressing VEGF or bFGF, or both, elicit a similarly durable angiogenic response.
The synergistic effect of VEGF and bFGF on vascular densities was most pronounced in the scar region, where a progressive increase from control to unmodified BMC to bFGF, VEGF, and VEGF and bFGF-expressing BMC was noted. The number of copies of bFGF mRNA in the groups transfected with this plasmid was roughly equivalent to the number of copies of VEGF mRNA in VEGF-transduced groups, but similar levels of expression of bFGF appeared to induce less of an increase in vascular densities than that of VEGF.
In the control rats, regional perfusion was lower in the scar than in the border zone and normal myocardium, as would be anticipated. A similar pattern was noted in the BMC group. In contrast, myocardial perfusion was greater in the border zone of the BMC+VEGF and BMC+VEGF+bFGF hearts than in either the scar or the normal myocardium. Vascular densities followed a similar pattern. That may reflect the localization of new vessel formation, developing from the border zone and extending toward the scar, where the angiogenic stimulus was centered.
Two and 4 weeks after cell transplantation, rats transplanted with unmodified BMC had greater LVEF than controls, consistent with the findings of Tomita and coworkers [14] at 8 weeks. However, because of the small number of rats in each group, these differences did not reach statistical significance. The addition of a VEGF or bFGF transgene, or both, resulted in significantly greater LVEF at both 2 and 4 weeks than the BMC or control groups. The LVEF was stable from 2 weeks to 4 weeks, but our study did not include later timepoints to determine whether the functional benefit of cell-based VEGF and bFGF gene transfer was durable beyond this point.
We did not evaluate the effect of the VEGF and bFGF transgenes on the survival of the transplanted BMC in this study. However, in previous studies, we have noted that expression of a VEGF transgene reduced apoptosis and increased the survival of transplanted heart cells and skeletal myoblasts at 1 week from approximately 30% to 50% [17]. We also noted that the simultaneous expression of both VEGF and insulin-like growth factor-1, or VEGF or insulin-like growth factor-1 alone in transplanted BMC had a synergistic effect on myogenesis, enhancing transplanted cell survival and reducing apoptosis [11]. We would anticipate, therefore, that BMC survival was increased in our current study in at least the BMC+VEGF+bFGF and BMC+VEGF groups, but cannot comment on the effect of bFGF alone.
While these types of multimodal cell-based gene therapeutic strategies may significantly enhance the benefit of cell transplantation in hearts with ischemic cardiomyopathy, the addition of complementary transgenes, cell types, and other reparative strategies may still be required to achieve complete myocardial repair after infarction. One such potential intervention is angiogenic pretreatment of the scar before cell implantation. Yamamoto and colleagues [18] have reported that prevascularization of a rat myocardial infarction by slow-release bFGF protein resulted in greater maximal elastance than either bFGF or cell transplantation alone. Sakakibara and associates [19] also noted greater cell survival with prevascularization than without. Sato and colleagues [20, 21] reported that intracoronary bFGF delivery results in significant improvement in collateralization and regional and global function in infarcted porcine hearts and also that intracoronary VEGF165 infusion improved collateralization and increased myocardial blood flow in the same model. Retuerto and associates [22] injected infarcted rat hearts with an adenoviral vector carrying VEGF121 before fetal cardiomyocyte transplantation, and noted greater exercise capacity and an increased cell survival index compared with pretreatment with empty vectors or saline. Scar pretreatment may thus extend the efficacy of cell-based gene therapeutic approaches to myocardial repair.
The results of our study must be tempered by the lack of statistical significance of some trends that we observed. Despite using a total of 109 rats in this experiment (75 recipients, 25 donors, and 9 replacements for rats that died after infarct creation), the division of these rats into five groups and four timepoints for evaluation resulted in only a small number of rats in each combination of group and timepoint. Thus, while vascular densities and regional perfusion in the border zone were significantly greater in the BMC+VEGF+bFGF rats than in the BMC+bFGF group, differences between the BMC+VEGF+bFGF and BMC+VEGF groups did not reach statistical significance, nor did those in the scar itself. However, our data were internally consistent, with trends favoring the BMC+VEGF+bFGF group over both the BMC+VEGF and BMC+bFGF rats at all timepoints for all variables studied. Although a greater number of rats would have been desirable, it seems reasonable to conclude, in light of the known biology of VEGF and bFGF, that these two transgenes acted synergistically in the current study.
In conclusion, our data demonstrate that VEGF and bFGF transgenes are transiently expressed by transplanted BMC, resulting in significantly increased vascular densities and regional perfusion in the scar and border zone of an infarcted heart, compared with unmodified BMC or controls. There was a powerful synergistic effect of VEGF and bFGF on this angiogenic response. In addition, LVEF was significantly increased by expression of VEGF or bFGF, compared with unmodified BMC or controls. Although the synergism between VEGF and bFGF significantly increased the angiogenic effect of BMC transplantation, it was still insufficient to normalize myocardial perfusion or function.
|
Acknowledgments |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
|
References |
|---|
|
TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
|---|
Related Article
This article has been cited by other articles:
|
|
 |
|
  S.-H. Li, T. Y.Y. Lai, Z. Sun, M. Han, E. Moriyama, B. Wilson, S. Fazel, R. D. Weisel, T. Yau, J. C. Wu, et al. Tracking cardiac engraftment and distribution of implanted bone marrow cells: Comparing intra-aortic, intravenous, and intramyocardial delivery. J. Thorac. Cardiovasc. Surg., May 1, 2009; 137(5): 1225 - 33.e1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Angoulvant, S. Fazel, R. D. Weisel, T. Y.Y. Lai, P. W. Fedak, L. Chen, S. Rafati, C. K. Seneviratne, N. Degousee, and R.-K. Li Cell-based gene therapy modifies matrix remodeling after a myocardial infarction in tissue inhibitor of matrix metalloproteinase-3-deficient mice. J. Thorac. Cardiovasc. Surg., February 1, 2009; 137(2): 471 - 480. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Spiegelstein, C. Kim, Y. Zhang, G. Li, R. D. Weisel, R.-K. Li, and T. M. Yau Combined transmyocardial revascularization and cell-based angiogenic gene therapy increases transplanted cell survival Am J Physiol Heart Circ Physiol, December 1, 2007; 293(6): H3311 - H3316. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Feng and F. W. Sellke Invited commentary Ann. Thorac. Surg., March 1, 2007; 83(3): 1119 - 1120. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
