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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    • "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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    ABSTRACT: Blood vessel growth plays a key role in regenerative medicine, both to restore blood supply to ischemic tissues and to ensure rapid vascularization of clinical-size tissue-engineered grafts. For example, vascular endothelial growth factor (VEGF) is the master regulator of physiological blood vessel growth and is one of the main molecular targets of therapeutic angiogenesis approaches. However, angiogenesis is a complex process and there is a need to develop rational therapeutic strategies based on a firm understanding of basic vascular biology principles, as evidenced by the disappointing results of initial clinical trials of angiogenic factor delivery. In particular, the spatial localization of angiogenic signals in the extracellular matrix (ECM) is crucial to ensure the proper assembly and maturation of new vascular structures. Here, we discuss the therapeutic implications of matrix interactions of angiogenic factors, with a special emphasis onVEGF, as well as provide an overview of current approaches, based on protein and biomaterial engineering that mimic the regulatory functions of ECM to optimize the signaling microenvironment of vascular growth factors.
    Frontiers in Bioengineering and Biotechnology 04/2015; 3(45). DOI:10.3389/fbioe.2015.00045
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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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    ABSTRACT: Rapid vascularisation of tissue-engineered osteogenic grafts is a major obstacle in the development of regenerative medicine approaches for bone repair. Vascular endothelial growth factor (VEGF) is the master regulator of vascular growth. We investigated a cell-based gene therapy approach to generate osteogenic grafts with an increased vascularization potential in an ectopic nude rat model in vivo, by genetically modifying human bone marrow-derived stromal/stem cells (BMSC) to express rat VEGF. BMSC were loaded onto silicate-substituted apatite granules, which are a clinically established osteo-conductive material. Eight weeks after implantation, the vascular density of constructs seeded with VEGF-BMSC was 3-fold greater than with control cells, consisting of physiologically structured vascular networks with both conductance vessels and capillaries. However, VEGF specifically caused a global reduction in bone quantity, which consisted of thin trabeculae of immature matrix. VEGF did not impair BMSC engraftment in vivo, but strongly increased the recruitment of TRAP- and Cathepsin K-positive osteoclasts. These data suggest that VEGF over-expression is effective to improve the vascularization of osteogenic grafts, but also has the potential to disrupt bone homoeostasis towards excessive degradation, posing a challenge to its clinical application in bone tissue engineering.
    Biomaterials 04/2013; 34(21). DOI:10.1016/j.biomaterials.2013.03.040 · 8.56 Impact Factor
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    • "Cells were stained with fluorescently labeled specific antibodies against human CD31, CD34, CD73, CD90 (BD Biosciences), VEGFR2 (R&D Systems, Abingdon, UK), CD105 (AbD Serotec, Dü sseldorf, Germany), and troponin I (Millipore, Zug, Switzerland) or with appropriate isotype control antibodies (BD Biosciences) according to manufacturers' instructions and analyzed with a FACS-Calibur Flow Cytometer (BD Biosciences). Retrovirus-encoded CD8a was detected with a specific mouse monoclonal antibody to rat CD8a (clone OX-8; BD Biosciences) as previously described (Wolff et al., 2012). Data were analyzed using FlowJo software (Tree Star, Ashland, OR). "
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    ABSTRACT: Vascular Endothelial Growth Factor (VEGF) can induce normal angiogenesis or the growth of angioma-like vascular tumors depending on the amount secreted by each producing cell, as it remains localized in the microenvironment. In order to control the distribution of VEGF expression levels in vivo, we recently developed a high-throughput FACS-based technique to rapidly purify transduced progenitors that homogeneously express a specific VEGF dose from a heterogeneous primary population. Here we tested the hypothesis that cell-based delivery of a controlled VEGF level could induce normal angiogenesis in the heart, while preventing the development of angiomas. Freshly isolated human adipose-tissue stem cells (ASC) were transduced with retroviral vectors expressing either rat VEGF linked to a FACS-quantifiable cell-surface marker (a truncated form of CD8), or CD8 alone as control (CTR). VEGF-expressing cells were FACS-purified to generate populations producing either a specific VEGF level (SPEC) or uncontrolled heterogeneous levels (ALL). Fifteen nude rats underwent intra-myocardial injection of 107 cells. Histology was performed after 4 weeks. Both the SPEC and ALL cells produced a similar total amount of VEGF and both cell types induced a 50%-60% increase in both total and perfused vessel density compared to CTR cells, despite very limited stable engraftment. However, homogeneous VEGF expression by SPEC cells induced only normal and stable angiogenesis. Conversely, heterogeneous expression of a similar total amount by the ALL cells caused the growth of numerous angioma-like structures. These results suggest that controlled VEGF delivery by FACS-purified ASC may be a promising strategy to achieve safe therapeutic angiogenesis in the heart.
    Human Gene Therapy 10/2012; 23(5):346-56. DOI:10.1089/hgtb.2012.032 · 3.76 Impact Factor
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