FACS‐purified myoblasts producing controlled VEGF levels induce safe and stable angiogenesis in chronic hind limb ischemia

Journal of Cellular and Molecular Medicine (Impact Factor: 4.01). 12/2011; 16(1):107 - 117. DOI: 10.1111/j.1582-4934.2011.01308.x


We recently developed a method to control the in vivo distribution of vascular endothelial growth factor (VEGF) by high throughput Fluorescence-Activated Cell Sorting (FACS) purification of transduced progenitors such that they homogeneously express specific VEGF levels. Here we investigated the long-term safety of this method in chronic hind limb ischemia in nude rats. Primary myoblasts were transduced to co-express rat VEGF-A164 (rVEGF) and truncated ratCD8a, the latter serving as a FACS-quantifiable surface marker. Based on the CD8 fluorescence of a reference clonal population, which expressed the desired VEGF level, cells producing similar VEGF levels were sorted from the primary population, which contained cells with very heterogeneous VEGF levels. One week after ischemia induction, 12 × 106 cells were implanted in the thigh muscles. Unsorted myoblasts caused angioma-like structures, whereas purified cells only induced normal capillaries that were stable after 3 months. Vessel density was doubled in engrafted areas, but only approximately 0.1% of muscle volume showed cell engraftment, explaining why no increase in total blood flow was observed. In conclusion, the use of FACS-purified myoblasts granted the cell-by-cell control of VEGF expression levels, which ensured long-term safety in a model of chronic ischemia. Based on these results, the total number of implanted cells required to achieve efficacy will need to be determined before a clinical application.

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Available from: Andrea Banfi
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    • "Using gene delivery systems such as retrovirally transduced myoblasts (Carmeliet, 2000; Lee et al., 2000), adeno-and adeno-associated viral vectors (Pettersson et al., 2000; Sundberg et al., 2001; Karvinen et al., 2011), and plasmid DNA (Isner et al., 1996; Schwarz et al., 2000), it was shown that uncontrolled VEGF expression induces the growth of vascular tumors (hemangiomas) in skeletal muscle (Springer et al., 1998), myocardium, and other tissues. However, we found evidence that VEGF does not have an intrinsically steep dose–response curve in vivo, but rather that the dose delivered must be controlled at the microenvironmental level (Ozawa et al., 2004; Von Degenfeld et al., 2006; Misteli et al., 2010; Melly et al., 2012; Wolff et al., 2012; Mujagic et al., 2013). In fact, due to the ECM-binding of VEGF, different GF concentrations remain tightly localized after secretion and a few " hotspots " of high expression can cause angioma growth even if the total dose is rather low. "
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    • "All the antibodies were used at a dilution of 1:50, except CD105-FITC, which was used at 1:20. CD8a expression was assessed by staining with a FITC-conjugated anti-rat CD8a antibody (clone OX-8; BD Pharmingen , San José, CA, USA), using previously optimized staining conditions [22] "
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    • "Our goal was to combine cell-based gene therapy and cardiac tissue engineering toward generating a fully viable cardiac patch with intrinsic vascularization potential. To this end, we took advantage of a FACS-based technique we recently developed to rapidly purify homogeneous populations of transduced cells producing a defined level of VEGF, to reliably induce robust and normal angiogenesis [27] [28]. This strategy is not directly applicable to cardiomyocytes, because they cannot be sufficiently expanded in vitro to allow efficient transduction and FACS-purification. "
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