Vascular plasticity and target for therapy in Duchenne muscular dystrophy
Kennedy, Donna
Kennedy, Donna
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Publication Date
2023-06-13
Type
Thesis
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Abstract
Angiogenesis is the growth of new blood vessels from pre-existing vessels, necessary for the growth and maintenance of homeostasis. Duchenne muscular dystrophy (DMD) is a debilitating disease of the musculoskeletal system. Altered vascularisation has been reported as a contributing factor in the disease pathology of DMD. In this study, rarefaction in capillary density and capillary to fiber ratio was reported in mdx mice. Similarly, significant reductions in vascular endothelial growth factor (VEGF) protein level and a blunting in transcript levels of VEGF receptors 1 and 2 were observed, indicating disruption to the VEGF/VEGFR pathway. VEGF-A is a potent angiogenic and myogenic factor, which has been shown to be a potential therapeutic to ameliorate symptoms of DMD. VEGF-A overexpression has been associated with the production of aberrant, leaky vascularisation, therefore co-delivery with another growth factor such as fibroblast growth factor (FGF) may remedy this. We developed stable transfected myoblast cell lines which expressed both VEGF-A alone and co-expressed VEGF-A/FGF-4, named Myo-VEGF-GFP and Myo-FGF4-VEGF. ELISA and RT-qPCR reported potent VEGF-A secretion from both cell lines. In vitro, the myogenic and angiogenic activity of these cells were examined using immunostaining and endothelial cell angiogenic assays. Myo-FGF4-VEGF cells exhibited increased rates of differentiation compared to control cell lines, while conditioned media from VEGF-A secreting cells resulted in increased migration, proliferation, and tube formation capabilities of C166 endothelial cells. In vivo, we investigated the angiogenic potential of these VEGF-A secreting myoblasts using the chick chorioallantoic membrane (CAM) assay. Conditioned media from VEGF-A secreting cells, as well as the cells themselves, resulted in a significant increase in angiogenic response in the CAM assay. Finally, using intravital fluorescent microscopy (IVFM) and Hoechst labelled erythrocytes we established a novel method to measure haemodynamic parameters in the CAM microcirculation. Conditioned media from VEGF-A secreting myoblasts resulted in a significant increase in erythrocyte velocity, functional capillary density and capillary perfusion in the CAM. The stable transfected myoblast established herein, provide sustained VEGF-A and FGF-4 release over a continuous period of time. The findings revealed in this study potentially provide broad implications in the field of therapeutic angiogenesis. Furthermore, the manipulation of these cells in higher-level animals may prove highly beneficial in the amelioration of vascular abnormalities and disease pathology in muscle diseases such as DMD.
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NUI Galway