Original articleCombining pharmacological mobilization with intramyocardial delivery of bone marrow cells over-expressing VEGF is more effective for cardiac repair
Introduction
The malleable nature of BMC with multilineage potential has been exploited for their ability to achieve cardiomyocyte phenotype for heart muscle regeneration [1], [2]. The number of BMC mobilized intrinsically in response to cardiac injury is too small to fully replenish the lost cardiomyocytes in the absence of an outside intervention. Pharmacological mobilization using various cytokines and growth factors egress stem cells from their bone marrow (BM) niches and shifts their homeostatic balance favorably in the peripheral circulation. The blood borne stem cells then home on to injured myocardium, enhance cardiac tissue regeneration and improve cardiac function after MI [3], [4].
Granulocyte colony-stimulating factor (G-CSF) and stem cell factor (SCF) are regulators of granulopoiesis and critically regulate proliferation, differentiation, and survival of myeloid progenitor cells [5]. They are angiogenic cytokines and distinctly enhance egress of hematopoietic stem cells (HSC) into the peripheral blood circulation [6] and engraftment following non-myoablative total-body irradiation [7].
Intramyocardial injection of stem cells from skeletal muscle and BM origin, with or without genetic modulation to over-express angiogenic growth factors lead to better prognosis in animal models [8], [9] In a recent report, Matsumoto et al. [10] have shown that transplantation of VEGF over-expressing BM derived MSC could enhance the cardioprotective effects of MSC, followed by angiogenic effects in salvaging host myocardium. A more interesting approach was adopted by Kawamoto et al. [32] who delivered VEGF2 gene by intramyocardial injection of adenoviral vector encoding for VEGF2 together with cytokine-induced mobilization of BMC. The synergistic effect of the combined therapy approach showed that mobilization of endothelial progenitor cells with cytokines potentiated angiogenic effects of VEGF2 gene therapy and enhanced their engraftment in the ischemic myocardium. Unlike Kawamoto's approach, our study examines intramyocardial delivery of BMC over-expressing hVEGF165 rather than direct intramyocardial viral vector delivery of VEGF, and combined with pharmacological mobilization of BMC. By tagging the intramyocardially transplanted and mobilized BMC with two different fluorescent dyes, we studied the fate of these cells in the infarcted myocardium. We hypothesized that the combined therapeutic consequence of intramyocardially delivered BMC overexpressing VEGF165 is amplified by increasing the supply of circulating progenitor cells via pharmacological mobilization by elevated levels of G-CSF and SCF.
Section snippets
Adenovirus vector propagation and purification
Adenoviral vectors carrying hVEGF165 gene (Ad-hVEGF165) or without hVEGF165 (Ad-Null) were provided by Dr. Ruowen Ge (Angiogenesis Laboratory, National University of Singapore). The vectors were propagated in 293 cells and purified on CsCl2 gradient. For gene transduction, MSC at 80% confluence were exposed to respective virus (1 × 109 particles/ml) for 8 hours followed by 24-hour culture in DMEM medium without viral vector. The transduction procedure was repeated three times to achieve optimal
Release of BMC into peripheral blood circulation
G-CSF and SCF caused a marked increase in the release of HSC into peripheral blood circulation. SCF is a hematopoietic cytokine which binds to c-kit receptor [18]. Fig. 1 shows the expression of CD34, Gr1, ckit and Sca-1 positive cells from the vehicle treated peripheral blood (A–D), and cytokine treated peripheral blood (E–H) on day 7. These data indicate that cytokine treatment gave significant increase in granulocytes (Gr1) and c-kit+ cells in peripheral blood.
Myocardial regeneration and distribution of c-Kit, BrdU and Ki67 positive cells
The differences in the effect
Discussion
The rationale of this study was that combined effect of cytokines with local administration of MSC over-expressing hVEGF165 would be superior to either of the strategies involved alone in repairing MI. Overall, this study demonstrates significantly enhanced BMC mobilization and endothelial cell proliferation, more pronounced angiogenic response, reduced tissue damage, and improved cardiac function following combined therapy of cytokines and delivery of MSC over-expressing hVEGF165.
The complex
Acknowledgments
This work was supported by NIH grants HL-081859-01 (Y. Wang) and HL-74272, HL080686 (M. Ashraf).
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