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Ann Thorac Surg 2007;83:640-646
© 2007 The Society of Thoracic Surgeons
Division of Cardiovascular Surgery, Toronto General Hospital, University Health Network, Department of Surgery, University of Toronto, and Heart and Stroke Foundation/Richard Lewar Centre of Excellence, Toronto, Ontario, Canada
Accepted for publication September 12, 2006.
* Address correspondence to Dr Yau, Division of Cardiovascular Surgery, Toronto General Hospital, 4N-470, 200 Elizabeth St, Toronto, ON M5G 2C4, Canada. (Email: terry.yau{at}uhn.on.ca).
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Abstract |
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METHODS: Lewis rats underwent myocardial cryoinjury 3 weeks before transplantation with heart cells (HC [a mixed culture of cardiomyocytes, smooth muscle cells, endothelial cells, and fibroblasts]), VEGF-transfected heart cells (HC+), skeletal myoblasts (Sk), VEGF-transfected skeletal myoblasts (Sk+), or medium (control) (n = 3 per group x 5 groups x 5 timepoints). Three days, 1 week, 2 weeks, 4 weeks, and 6 months after transplantation, hearts were excised and the scar, border zone, and normal myocardium evaluated for angioma or sarcoma formation, and vascular density quantitated.
RESULTS: Vascular densities were lowest in controls, intermediate in HC and Sk (p < 0.05) and highest in HC+ and Sk+ (p < 0.05). Densities were highest in the border zone, intermediate in normal myocardium, and lowest in the scar (p < 0.05), peaking in the border zone of HC+ and Sk+ hearts at 4 weeks. At 6 months, densities were greater in HC and Sk than controls (p < 0.05), and higher in Sk+ than Sk (p < 0.05), but HC+ and HC were similar. There was no evidence of angioma or sarcoma formation at any timepoint.
CONCLUSIONS: Angiogenesis induced by HC and Sk transplantation lasts at least 6 months and is increased by transfection with VEGF without apparent oncogenicity. The durability of the VEGF effect on vascular densities may be greater with Sk.
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Introduction |
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We have observed that VEGF transgene expression in these transplanted cells is limited to the scar and border zone, and lasts approximately 4 weeks [9], while upregulating the VEGF receptors flk-1 and flt-1 through a paracine effect on donor cells [10]. Ex vivo modification of cells before transplantation may therefore have the potential to enhance survival of the transplanted cells and modify their effect on myogenesis, angiogenesis, or matrix remodeling. However, the durability of the angiogenic response to this transient VEGF overexpression is unknown. Rapid induction but rapid regression of vascular ingrowth would significantly limit the long-term utility of cell-based angiogenic gene therapy. Conversely, excessive stimulation may result in formation of angiomas or sarcomas, as has been previously reported as a result of long-term VEGF expression [11].
In this study, we hypothesized that cell transplantation alone would increase vascular densities in the scar and border zone, but that transient expression of a VEGF transgene in these cells would increase both the magnitude and the duration of this angiogenic effect. We also hypothesized that this angiogenic response would not result in observable oncogenicity during the 6-month duration of this study. Finally, we evaluated the effect of two different cell types, to determine whether there were cell-type–specific differences in either the durability or the safety of this effect.
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Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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A large transmural scar was created in the left ventricular (LV) free wall of rat hearts by a cryoinjury technique, as previously described [8]. Briefly, through a left lateral thoracotomy, the LV free wall was exposed and cryoinury performed by 12 1-minute applications of an 8 x 10 mm elliptical metal probe cooled to –196°C by immersion in liquid nitrogen. After recovery, the cryoinjured rats were randomly divided into five experimental groups: control, injected with medium without cells, transplantation with a mixed culture of unmodified heart cells (HC), transplantation with VEGF-transfected HC (HC+), transplantation with unmodified skeletal myoblasts (Sk), or transplantation with VEGF-transfected Sk (Sk+) (n = 3 per group x 5 groups x 5 timepoints = 75 recipient rats total).
Cell Isolation and Culture
A mixed primary culture of cardiomyocytes, smooth muscle cells, endothelial cells, and fibroblasts was isolated from the left ventricles of donor rats as previously described [8]. The cultured cells were depleted of fibroblasts by a preplating technique and then maintained in Iscove’s modified Dulbecco’s medium containing 10% fetal bovine serum for 5 to 7 days. In a subset of plates, 70% of cells stained positively for myosin heavy chain and were assumed to be cardiomyocytes, 15% of cells stained positively for alpha-smooth muscle actin and were assumed to be smooth muscle cells, and 11% of cells stained positively for factor VIII and were assumed to be endothelial cells. The remainder of the cells were assumed to be fibroblasts.
Primary skeletal myoblasts were isolated and cultured by a modified single-muscle fiber culture technique [9, 10, 12]. Briefly, muscle (3 g) from the quadriceps femoris muscle of adult male donor Lewis rats was digested with protease and type I collagenase (Sigma, St. Louis, Missouri) before isolation and resuspension of single, intact muscle fibers in Iscove’s modified Dulbecco’s medium containing 10% fetal bovine serum. After preplating, single skeletal muscle fibers were plated onto laminin-coated plates (Becton Dickinson, Bedford, Massachusetts). Skeletal myoblasts dissociated from the muscle fibers, attached to the plate, and were allowed to proliferate for 48 to 72 hours as the original muscle fibers underwent cell death and lysis. The skeletal myoblasts were maintained in Iscove’s modified Dulbecco’s medium containing 10% fetal bovine serum for 5 to 7 days and transfected before fusion and myotube formation.
Cell Transfection
Skeletal myoblasts and heart cells were transfected for 24 hours in 100-mm dishes at 60% to 70% confluence. Cells were transfected ex vivo, by Effectene (Qiagen, Mississauga, Ontario), a nonliposomal lipid formulationwith a plasmid encoding VEGF165 (pCEP4-VEGF), as previously described [8–10]. Transfection efficiencies were monitored in a subset of plates by cotransfection with pEGFP-N2 (BD Biosciences, Palo Alto, California), expressing green fluorescence protein. Transfection efficiencies for both cell types were approximately 30%.
Bromodeoxyuridine Prelabeling
One of every four plates of cells was prelabeled with bromodeoxyuridine 2 days before transplantation and 1 day before transfection [8–10]. A monoclonal antibody directed against bromodeoxyuridine was used to identify the transplanted cells within the recipient hearts.
Cell Transplantation
Rats underwent cell transplantation 3 weeks after LV cryoinjury. Donor male rat mortality after cryoinjury was less than 20%. Cells were detached from culture dishes with trypsin, centrifuged, and resuspended in serum-free medium. Under general anesthesia, rat hearts were exposed through a midline sternotomy. Three million cells in 0.05 mL serum-free medium, or the same volume of medium without cells, were injected at multiple points into the center of the LV scar with a 28G needle.
Rats were sacrificed and the hearts excised 3 days, 1 week, 2 weeks, 4 weeks, or 6 months after cell transplantation. The atria and the right ventricular free wall were excised, leaving the LV, which was divided into the scar zone (transmural scar), the border zone (partial-thickness scar), and normal myocardium.
Immunohistochemical Assays and 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 (Sigma-Aldrich Canada Limited, Oakville, Canada) or with antibodies against factor VIII, to facilitate quantitation of vascular density. The number of vessels per high power field (0.2 mm2) 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 employed for analysis.
Statistical Analysis
To evaluate the determinants of vascular density, we analyzed the effect of group, time, and location by analysis of variance (ANOVA) using SAS statistical software (SAS Institute, Cary, North Carolina). Both main effects and interactive effects were evaluated. Nonsignificant (p > 0.05) interactive effects were discarded, and the final model included only the statistically significant main and interactive effects. When the ANOVA F-value was significant, specific differences between groups or timepoints were identified by Duncan’s multiple range test. To statistically evaluate the effect of VEGF transfection and cell type rather than group assignment in general, a second analysis was performed on only the four cell-transplanted groups, evaluating the effects of VEGF transfection (yes versus no), cell type (HC versus Sk), time, and location by ANOVA.
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Results |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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Effect of cell transplantation alone
Vascular densities in the scar and border zone of control rat hearts injected only with medium were extremely low and did not vary significantly with time (Fig 1A and 1B). In hearts transplanted with unmodified HC or Sk, vascular densities were increased by approximately 60% in the scar compared with controls, and densities in the border zone were approximately doubled (p < 0.05). Densities in the normal myocardium were unaffected by cell transplantation at any time.
Effect of transfection with VEGF
Transfection with VEGF resulted in dramatically increased vascular densities in the HC+ and Sk+ rats compared with the HC and Sk transplanted groups or controls (p < 0.05). Peak vascular densities in both the scar and the border zone of HC+ and Sk+ hearts were approximately double those in HC and Sk rats, and approximately fourfold greater than those in control hearts (p < 0.05). Densities in normal myocardium, however, were unaffected by HC+ or Sk+ transplantation.
Effect of time
There was no significant time-dependent variation in vascular densities in the HC and Sk rats, and the differences seen between HC and Sk rats versus control rats were persistent to the 6-month timepoint. In contrast to the effect of cell transplantation alone, in which there was little variation with time, there was a strong time-dependent effect of VEGF transfection, with vascular densities in the HC+ and Sk+ groups increasing steadily from 3 days to 4 weeks before declining at 6 months (p < 0.05).
Six months after cell transplantation, vascular densities in the scar of Sk+ hearts were still significantly greater than those in any other group (p < 0.05 versus HC+, HC, Sk, control). Densities in the HC+, HC, and Sk rats were still greater than control (p < 0.05), but the difference between HC+ and HC was not statistically significant. At 6 months, vascular densities in the border zone of all cell-transplanted groups remained significantly higher than control hearts (p < 0.05), but there was no significant difference between the four cell-transplanted groups. Densities in the normal myocardium of all 5 groups were similar.
Effect of cell type
In contrast to the dramatic effects of VEGF transfection, time, or zone, vascular densities were not significantly influenced by cell type (p = 0.5). Both HC and Sk induced similar degrees of angiogenesis after transplantation, and both had similar increases in effect with VEGF transfection.
Oncogenicity
Histologic examination of the scar and border zone in control rats revealed a hypocellular scar with very few vascular spaces and a thinned scar region consisting predominantly of collagen and fibroblasts (Fig 2).
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In the HC+ and Sk+ transplanted rats, donor cells were again clearly visible in the transplanted scar. Qualitatively, the degree of engraftment appeared to be somewhat increased, although this was not quantitated. Vascular densities were obviously greater, particularly in the border zone at the 2-week and 4-week timepoints, as quantitated above. Again, there was no evidence of angioma or sarcoma formation at any timepoint evaluated.
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Comment |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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We have previously reported that the angiogenic response of cell transplantation can be augmented by the implantation of VEGF-transfected cells [8]. Expression of VEGF, while transient and localized [9], induces sequential upregulation of the VEGF receptors flk-1 and flt-1 in host cells through a paracrine effect [10], and leads to enhanced angiogenesis. Transplanted cell survival was also significantly increased by expression of the VEGF transgene, and may have been due to the induction of angiogenesis and vasculogenesis in an ischemic environment [19]. Smooth muscle cells expressing insulin-like growth factor-1 demonstrated induction of VEGF expression, greater angiogenesis, and reduced apoptosis [20]. We have evaluated the combination of VEGF and insulin-like growth factor-1 transgenes in an attempt to maximize their effect on cell survival and ventricular function. We noted that the simultaneous expression of both VEGF and insulin-like growth factor-1 transgenes in transplanted bone marrow cells had a synergistic effect on myogenesis, further enhancing transplanted cell survival, LV ejection fraction, and cardiac contractile protein content [21].
Our current study demonstrates that the angiogenic response to cell transplantation can be significantly enhanced in both the scar and the border zone by the expression of a VEGF transgene, and that vascular densities peak 4 weeks after implantation. Whereas transgene expression is transient, the angiogenic effect of this gene expression is more durable, persisting to at least 6 months, the last timepoint at which rats were evaluated in this study. Vascular densities from 3 days to 4 weeks were similar between the HC transplanted and the Sk transplanted rats, indicating that the proangiogenic effect of the VEGF transgene was not limited to either cell type. Six months afterward, vascular densities were still greater in the Sk+ group than in all other groups, indicating a durable response. Although vascular densities in the HC+ group were still elevated over baseline, there was not a significant difference between HC+ and HC, perhaps owing to the limited number of rats evaluated at the 6-month timepoint. By 6 months, however, cell engraftment is complete, and the survival of any viable hibernating host cardiomyocytes in the injured region will also have been determined. The minor differences between HC and Sk may not translate into functional differences 6 months after transplantation.
Vascular densities in the cell-transplanted and VEGF-transfected cell-transplanted rats were greater in the border zone than in the center of the scar. It has previously been shown that VEGF-transfected Sk significantly enhance vascular density at 3 months in the peri-infarct region of pig hearts [22]. Cell engraftment was identified almost exclusively in the scar, where the cells had been injected. Therefore, the treatment produced an effect at a distance, confirming the paracrine influence of cell and transfected cell transplantation. This angiogenic response could have been due to the release of a variety of proteins or the remodeling of the extracellular matrix.
Prolonged angiogenic stimulation has been associated with angioma formation [11, 23]. Using our current technique of cell transfection, VEGF expression by the transplanted cells is transient and localized [9]. This transient expression did not result in formation of angiomas or any evidence of local accumulation of proliferating cells, even 6 months after cell implantation. Similarly, Suzuki and colleagues [24] have previously reported that VEGF-transfected Sk expressed VEGF transiently, without angioma formation at 1 month after treatment. Thus, the addition of VEGF transfection did not lead to detectable oncogenicity after cell transplantation.
Establishing the safety and potential efficacy of cell-based gene therapy will require an extensive series of preclinical investigations. This study is only one of many evaluations that will be required before the clinical application of this therapy. However, capillary density may be the most important characteristic of this intervention. Extending the vascular network is the first requirement for successful angiogenesis. Other studies have already reported that angiogenic transgene expression can increase cell survival and LV function [19, 21].
This study has distinct limitations. The cryoinjury model is not clinically relevant, but provides a severe test for the angiogenic potential of gene-enhanced cell transplantation. The resultant scar may be most similar to the thinned, dilated scar of a LV aneurysm resulting from an infarct in a patient without preformed collaterals. In these patients, cell transplantation offers the promise of ventricular restoration that cannot be anticipated with protein or gene therapy alone. Determination of the significance and relevance of these findings will, however, require more extensive studies.
In summary, VEGF-transfected myocytes increased vascular densities more than cell transplantation did alone. The increase was greater in the border zone than in the center of the injured region and increased for 4 weeks after cell implantation. Six months later, the capillary density continued to be greater in the cell-transplanted groups than the control group, and VEGF transfection further increased capillary densities in the Sk+ group. Our experiments demonstrate that the expression of an angiogenic trangene further enhances the angiogenic effect of heart cell and skeletal myoblast transplantation. Further studies will be required to determine the optimal application of cell-based gene therapy for myocardial repair.
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Acknowledgments |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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References |
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TopAbstractIntroductionMaterial and MethodsResultsCommentAcknowledgmentsReferences |
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