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J Thorac Cardiovasc Surg 2004;127:535-540
© 2004 The American Association for Thoracic Surgery
Cardiopulmonary support and physiology |
a Evanston Northwestern Healthcare, Evanston, Ill, USA
b Northwestern University Medical School, Chicago, Ill, USA
c Weill Medical College of Cornell University, New York, NY, USA
d University of Illinois at Chicago, Chicago, Ill, USA
Received for publication December 9, 2002; revisions received April 21, 2003; accepted for publication June 5, 2003.
* Address for reprints: Todd K. Rosengart, MD, 2650 Ridge Ave, Burch 100, Evanston, IL 60201, USA
trosengart{at}enh.org
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Abstract |
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METHODS: New Zealand White rabbits received adenovirus (5 x 1010 particle units) encoding for vascular endothelial growth factor 121 (n = 14) or a control vector without a transgene (n = 13) or saline solution (n = 9) via direct myocardial injection. Fluorescent microsphere perfusion studies and histologic analyses were performed 4 weeks later. In a parallel study, exercise treadmill testing was performed to assess the functional effects of this therapy in Sprague-Dawley rats.
RESULTS: Microsphere assessment of myocardial perfusion in rabbits 4 weeks after adenovirus-encoding vascular endothelial growth factor administration was greater than that for rats injected with control vector without a transgene or saline solution (3.2 ± 0.5 vs 2.7 ± 0.7 and 2.4 ± 0.4, respectively; P < .03). The endothelial cell count per high power field was increased in animals injected with adenovirus-encoding vascular endothelial growth factor versus animals injected with control vector without a transgene or saline solution (147 ± 27 vs 123 ± 14 and 125 ± 16 cells, respectively), although this did not reach statistical significance (P = .12). Rats treated with adenovirus-encoding vascular endothelial growth factor also demonstrated prolonged exercise tolerance compared with rats injected with control vector without a transgene or saline solution (exhaustion time: 26 ± 5 minutes vs 19 ± 2 minutes and 20 ± 3 minutes, respectively; P = .006).
CONCLUSIONS: Adenovirus encoding–mediated transfer of vascular endothelial growth factor 121 induces an enhancement in regional perfusion in nonischemic myocardium that corresponds to changes in exercise tolerance. Adenovirus-encoding vascular endothelial growth factor therapy may be useful for inducing angiogenesis in the nonischemic state, such as for prophylactic therapy of early coronary artery disease.
Contrary to these suppositions, growth factor administration has been demonstrated to enhance tissue vascularization in several nonischemic models.10-15 Growth factors have also been successfully administered as a prophylactic therapy prior to vascular ligation.13-15 It is likely that this observed induction of angiogenesis in nonischemic tissues is made possible in part by the ability of growth factors to up-regulate the expression of their own receptor; for example, as in the case of vascular endothelial growth factor (VEGF) up-regulation of flk-1.16
It has not been previously determined whether the administration of angiogenic growth factors will cause a physiologically significant increase in perfusion to nonischemic tissues, as opposed to only increasing blood vessel number. Theoretically, such enhanced perfusion might result in improved functional capabilities of these tissues. Importantly, such findings would refute the existing presumption that up-regulation of receptor expression in ischemic tissues will limit angiogenic effects to these tissues following systemic growth factor administration. This study consequently investigates the effects of adenoviral-mediated transfer of VEGF121 cDNA to nonischemic myocardium and demonstrates enhanced myocardial perfusion and improved exercise tolerance in this setting.
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Materials and methods |
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500 ng VEGF detected per 1 x 1011 particle units (pu) of AdVEGF assayed in culture.
Perfusion studies (rabbit)
Male New Zealand White rabbits (average weight 3 kg; Covance Research Products, Denver, Pa) were treated in accordance with institutional animal care and use committee (IACUC) guidelines. The rabbits were sedated with subcutaneous ketamine (35 mg/kg) and xylazine (2 mg/kg). Following endotracheal intubation, general anesthesia was maintained with 3 L O2 and intravenous fentanyl (150 µg/kg). A left lateral thoracotomy was then performed, and 5 x 1010 pu of AdVEGF (n = 14), AdNull (n = 13), or saline solution (n = 9) was delivered via direct myocardial injection using a 27G tuberculin syringe at 4 sites on the anterior and posterior left ventricular walls at the midventricular level (25 µL per injection), encompassing the left ventricular free wall.
Four weeks later, a repeat thoracotomy was performed and 2 x 106 fluorescent 15-µm microspheres (Molecular Probes, Eugene, Ore) were injected via left atrial cannulation. After killing the animals under general endotracheal anesthesia, the left ventricle was sectioned into upper, middle, and apical thirds. The upper and middle sections (rings) were divided into septal, anterior, and posterior segments, while the apical section was preserved as 1 segment. Spheres were liberated from these segments by alkali digestion and quantified in a blinded fashion, as previously described.18 An index of perfusion was calculated as the ratio of the microspheres per gram of tissue extracted from each ventricular segment divided by the number of microspheres per gram of tissue extracted from the (untreated) upper septum, which served as an internal reference. A composite upper and lower ventricular perfusion index was calculated as the sum of the perfusion indices of the treated upper and middle rings, respectively.
Histologic evaluation (rabbit)
Three additional groups of rabbits underwent the administration of AdVEGF (n = 9), AdNull (n = 8), or saline solution (n = 7) as described above. Four weeks later, the animals were killed after a pentobarbital overdose (150 mg/kg IV), the hearts were prepared for histologic analysis in 10% neutral buffered formalin, embedded in paraffin, and sectioned. Endothelial cells were stained for 1 hour with a 1:100 dilution of biotinylated Griffonia simplicifolia lectin (Vector Labs, Burlingame, Calif). Stained regions were visualized using an avidin-biotin-peroxidase complex and diaminobenzidine substrate, as previously described. The mean endothelial cell count was calculated in a blinded fashion from the number of endothelial cells seen in 3 randomly selected high-power fields from these sections of the injected regions of the left ventricle and the (uninjected) upper and lower septum.
Exercise protocol (rat)
Male Sprague-Dawley rats (average weight 360 g; Harlan, Indianapolis, Ind) were treated in accordance with IACUC guidelines. Rats were administered five injections (20 µL) of AdVEGF or AdNull (1 x 1010 pu) or saline solution (n = 8 per group) in a manner analogous to that for the rabbit studies. Prior to injection, all animals were trained for 10 minutes on a multitrack small animal motorized treadmill (Model 42-15, Quinton, Bothell, Wash) at a constant speed of 15 m/min. Two weeks after surgery, the rats were exercised on the same treadmill according to a standardized protocol, as follows: 10 m/min for 5 minutes, 15 m/min for 5 minutes, and 25 m/min for the remainder of the experiment.19 Exhaustion was defined as the time point at which the animals rats sustained 10 falls per minute onto the electrified grid at the end of each running track.19
Statistical analysis
Data are expressed as means and standard deviations. Analysis of variance with Bonferroni correction was used to show significance for all analyses.
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Results |
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Rabbit myocardial histology
Mean endothelial cell number per microscopic field in the injected anterior and posterior midventricular segments was increased in animals injected with AdVEGF versus AdNull or saline solution (147 ± 27 vs 123 ± 14 and 125 ± 16 cells, respectively), although this did not reach statistical significance (P = .12; Figure 2, A). Consistent with the expectation of localized spread of the Ad vector beyond the area immediately surrounding the injection site, endothelial cell number in the noninjected lower septum also demonstrated a trend toward increased values in animals injected with AdVEGF versus AdNull or saline solution (156 ± 28 vs 112 ± 26 and 114 ± 18 cells, respectively; P = .13). Qualitative analysis of treated territories is remarkable for the lack of inflammation or infarction noted in these tissues (Figure 2, B, C). Histologic analysis in AdVEGF-treated animals furthermore suggests a predominant pattern of small-vessel development.
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Discussion |
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Although it cannot be ruled out that some degree of ischemia induced by surgical manipulation or other stresses did not play a role in enhancing perfusion in this study, the significantly greater changes in perfusion and exercise tolerance observed in the VEGF compared with the non-VEGF control groups suggest that this is not the case. Although it would be useful to determine whether substantial degrees of ischemia would have created an even greater enhancement of perfusion than that observed in this nonischemic model, the presence of different baseline levels of perfusion in ischemic versus nonischemic hearts unfortunately precludes a meaningful comparison of this parameter. Further, given the relatively high levels of perfusion in the nonischemic myocardium, it is not unexpected that the increases in perfusion noted in this study were only modest.
The observed increase in endothelial cell number in the present study, although not statistically significant, is consistent with improvements seen in previously reported results for nonischemic tissues.1,13,14 It is difficult to account for the relative discrepancy between the modest increase in endothelial cell number and relatively greater increases in physiologic parameters noted in these and other studies in ischemic models.1-6 Conceivably, these differences may be related in some way to the artifact of the "two-dimensionality" of histologic analyses (ie, many cells out of the microscopic plane are not visualized) and the potentially nonlinear relationship between new vasculature and the perfusion and enhancement of function potentially provided by these vessels.
It is interesting to note that endothelial cell number and perfusion to the ventricular septum were increased in the present study, despite the limiting of injections to the left ventricular free wall. This finding is consistent with a pattern of global improvement in ventricular function we have noted following direct myocardial injection of AdVEGF to the left ventricular free wall in a pacing model of cardiomyopathy20 and our prior observations of only partial localization of transgene expression following intramyocardial delivery.21-23 Localized angiogenic effects in treated areas may result in enhanced perfusion to neighboring territories through collateral flow, such as the septal branches of the left anterior descending, which in turn is situated in a treated myocardial territory. Alternatively, the septal tissue may be exposed to AdVEGF through the local diffusion of the aqueous vector solution through the myocardium or through transmyocardial vascular transport.
Data in the present study suggest but do not prove the likelihood of long-term persistence of the noted perfusion changes, nor do they prove a prophylactic capability for this therapy in preventing or minimizing myocardial infarction in the setting of a subsequent vascular occlusion. Given the fact that adenoviral-mediated VEGF expression is largely extinguished by 1 week after vector administration,12,21-23 however, the presence of increased perfusion at 4 weeks after vector administration suggests that these changes persist at least beyond the duration of VEGF expression. Our prior observations of neovasculature persistence at 90 days following AdVEGF administration in the rat fat pad and findings of a prophylactic effect in peripheral and myocardial ischemia models after vector administration12-15 similarly suggest that persistence of perfusion changes is possible, as do our recent findings in a prophylactic peripheral model 6 weeks after vector administration (unpublished data). It has been suggested that the administration of angiogenic cofactors such as angiopoietin-1 or platelet-derived growth factor might further stabilize and allow the persistence of a neovasculature.24,25 Alternatively, the presence of flow through newly created vessels might create sufficient impetus for persistence without such additional therapy. The findings of this study suggest that angiogenic therapy may be of benefit in treating tissues not traditionally considered to be ischemic, such as those neighboring an ischemic myocardial segment in patients with coronary artery disease, or in nonatherosclerotic conditions, such as ventricular hypertrophy or enlargement in the setting of valvular disease. These findings also suggest the possibility of creating tissues that may function in "hypernormal" modes; for example, when accelerated wound healing is required. Finally, given evidence of correlations between treadmill aerobic running capacity and left ventricular function in isolated heart preparations,26,27 the enhancement of exercise tolerance noted in the AdVEGF-treated "normal" animals may indicate that myocardial perfusion may be a limiting component in cardiac performance. We are aware that such treatment holds implications for improving athletic performance.
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Acknowledgments |
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Footnotes |
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References |
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  L. Ye, H. Kh. Haider, S. Jiang, R. S. Tan, R. Ge, P. K. Law, and E. K.W. Sim Improved angiogenic response in pig heart following ischaemic injury using human skeletal myoblast simultaneously expressing VEGF165 and angiopoietin-1 Eur J Heart Fail, January 1, 2007; 9(1): 15 - 22. [Abstract] [Full Text] [PDF]
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