Cellular, but not direct, adenoviral delivery of vascular endothelial growth factor results in improved left ventricular function and neovascularization in dilated ischemic cardiomyopathy
Arman Askari, MD*,
Samuel Unzek, MD ,
Corey K. Goldman, MD, PhD,
Stephen G. Ellis, MD, FACC*,
James D. Thomas, MD, FACC*,
Paul E. DiCorleto, PhD,
Eric J. Topol, MD, FACC* and
Marc S. Penn, MD, PhD, FACC*,*
* Department of Cardiovascular Medicine, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio, USA
Department of Cell Biology, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland, Ohio, USA
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Figure 1 Direct adenoviral injection or transplantation of cells expressing vascular endothelial growth factor (VEGF)-165 induces neovascularization. Eight weeks after myocardial infarction induced by left anterior descending coronary artery ligation, the peri-infarct zone was injected with either 1 x 107 pfu of adenoviral VEGF-165 (AdVEGF-165) (n = 4) or 1 million skeletal myoblast (SKMB) transfected with AdLuciferase (n = 7) or AdVEGF-165 (n = 6) in five equally divided injections. The vasculature was identified by von Willebrand factor immunohistochemistry, and vascular density was quantified throughout the infarct zone. Data represent mean ± SD. *p < 0.01 compared with SKMB transplantation alone. &p < 0.05 compared with AdVEGF injection.
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Figure 2 Vascular endothelial growth factor (VEGF)-165 induces neovascularization in the infarct zone. Representative sections of the infarct zone four weeks after injection into the peri-infarct zone of either 1 million skeletal myoblasts (SKMB) transfected with AdLuciferase (A, D), 1 x 107 pfu adenoviral VEGF-165 (AdVEGF-165) (B, E), or 1 million SKMB transfected with AdVEGF-165 (C, F) in five equally divided injections. (A, B, C) H & E staining and (D, E, F) immunohistochemistry for von Willebrand factor.
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Figure 3 Transplantation of vascular endothelial growth factor (VEGF)-165 expressing skeletal myoblasts (SKMB) significantly increases LV function. Eight weeks after myocardial infarction, the peri-infarct zone was injected with either saline (n = 7), 1 x 107 pfu of adenoviral VEGF-165 (AdVEGF-165) (n = 4), or 1 million SKMB transfected with AdLuciferase (n = 7) or AdVEGF-165 (n = 6) in five equally divided injections. Left ventricular function was quantified four weeks later by echocardiogram. Left ventricular function is presented as shortening fraction (%) relative to saline control. Data represent mean ± SEM. *p < 0.03 compared with saline control. &p < 0.05 compared with SKMB.
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Figure 4 Adenoviral vascular endothelial growth factor (AdVEGF-165) transfection does not alter skeletal myoblast (SKMB) engraftment. (A) Representative lanes from Western blot analyses of 50 µg of protein extract from whole hearts five days after sham operation (n = 3), or transplantation with 1 x 106 SKMB transfected with either AdLuciferase (n = 3) or AdVEGF-165 (n = 4) 18 h before transplantation using antibodies specific to either skeletal myocyte myosin heavy chain (MY-32) or the light chain of cardiac myosin. (B) Quantification of MY-32 bands from Western blot analysis in arbitrary units. Data represent mean ± SD. p = NS SKMB vs. SKMB + AdVEGF-165.
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Figure 5 Adenoviral vascular endothelial growth factor-165 (AdVEGF-165) injection results in increased TUNEL-positive cardiac myocytes in the infarct border zone. Representative TUNEL-stained (red) nuclei in sections from the peri-infarct zone four weeks after injection of (A) 1 million skeletal myoblasts (SKMB), (B) 1 x 107 pfu AdVEGF-165, or (C) 1 million SKMB transfected with 1 x 107 pfu AdVEGF-165 each in five equally divided injections. (D) Number of TUNEL-positive cells (white bars) and TUNEL-positive cardiac myocytes (black bars) per 100 nuclei as quantified in five high power fields ( 600 nuclei) from the infarct border zone per animal. Data represent mean ± SD. n = 4 animals per group. p < 0.01 direct viral injection versus transfected SKMB for both Luc and VEGF-165. No significant differences were observed between VEGF-165 and Luc within each delivery strategy.
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