Therapeutic benefits of angiogenetic gene-modified human mesenchymal stem cells after cerebral ischemia
Introduction
Intravenous injection of human mesenchymal stem cells (hMSCs) several hours after ischemia onset can reduce infarction size and improve functional outcome in rat cerebral ischemia models (Chen et al., 2001, Honma et al., 2006, Horita et al., 2006, Iihoshi et al., 2004, Li et al., 2002, Liu et al., 2006, Nomura et al., 2005). Although the detailed mechanisms of therapeutic benefits of MSCs injection remain unclear, angiogenesis is thought to be involved in neurological recovery after ischemia (Chen et al., 2003, Krupinski et al., 1994, Onda et al., 2007).
Vascular endothelial growth factor (VEGF) and angiopoietin 1 (Ang-1) are both important in angiogenesis (Carmeliet and Collen, 1997), but have different effects on vessel formation. VEGF has strong angiogenic effects in brain (Zhang and Chopp, 2002), limb (Baumgartner et al., 1998) and myocardial ischemia models (Kastrup, 2003). It is required to initiate the formation of immature vessel trunks (Carmeliet and Collen, 1997). Angiopoietin is involved in maturation, stabilization, and remodeling of vessels (Davis et al., 1996, Suri et al., 1996, Suri et al., 1998, Yancopoulos et al., 2000), and promotes angiogenesis in the brain (Onda et al., 2007, Ward and Lamanna, 2004). VEGF treatment alone to promote neovascularization can have deleterious effects. It is a potent enhancer of vascular permeability to blood plasma proteins which could contribute to brain edema after cerebral infarction (Bates et al., 2002, Wang et al., 1996). Ang-1 protects the peripheral vasculature from vascular leakage (Thurston et al., 1999) and thus might account for its anti-edematic effects following cerebral ischemia. We have recently demonstrated that intravenous infusion of hMSCs genetically modified to secrete Ang-1 has a modest neuroprotective effect in a rat cerebral ischemia model (Onda et al., 2007). The present study was designed to determine whether VEGF and the combined hypersecretion of VEGF and Ang-1 by hMSCs could influence angiogenesis and functional outcome in a rat stroke model.
Section snippets
Preparation of human mesenchymal stem cells
Human mesenchymal stem cells were processed for cell culture as previously described (Honma et al., 2006, Liu et al., 2006, Nomura et al., 2005). Briefly, human bone marrow from healthy adult volunteers was obtained by aspiration from the posterior iliac crest after informed consent was obtained; the subject's consent was obtained according to the Declaration of Helsinki, and this study was approved by the Institutional Review Board at Sapporo Medical University. Bone marrow was plated in 150 cm
Characteristics of primary and Ang-1, VEGF, and Ang-VEGF-hMSC gene-transduced hMSCs
Primary hMSCs were cultured as plastic adherent cells to subconfluency (see Materials and methods). Both flattened and spindle shaped-cells were present in the cultures. The hMSCs (Fig. 1A) had a CD34−, CD45−, SH2+ (CD105), and SH3+ (CD73) cell surface phenotype which is typical for hMSCs (Kobune et al., 2003). The genetically engineered cells (Ang-hMSCs, VEGF-hMSC, and Ang-VEGF-hMSC) showed a similar flattened and spindle shaped morphology in culture. Flow cytometric analysis of the Ang-hMSCs (
Discussion
A number of previous studies have demonstrated beneficial effects of hMSC transplantation in experimental cerebral ischemic models (Chen et al., 2001, Honma et al., 2006, Horita et al., 2006, Liu et al., 2006, Nomura et al., 2005, Ukai et al., 2007, Onda et al., 2007). The precise mechanisms of these effects are not clear, but angiogenesis is thought to be an important contributing factor (Onda et al., 2007). Both VEGF and Ang-1 contribute to vascular formation and maturation in an apparently
Acknowledgments
This work was supported in part by grants from the Japanese Ministry of Education, Science, Sports and Culture (16390414, 20390388), the National Multiple Sclerosis Society (U.S.A) (RG2135; CA1009A10), the National Institutes of Health (NS43432), and the Medical and Rehabilitation and Development Research Services of the Department of Veterans Affairs.
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