Abstract
Recent strategies to treat peripheral arterial disease (PAD) have focused on stem cell based therapies, which are believed to result in local secretion of vascular growth factors. Little is known, however, about the role of ischemic endogenous cells in this context. We hypothesized that ischemic muscle cells (MC) are capable of secreting growth factors that act as potent effectors of the local cellular regenerative environment. Both muscle and endothelial cells (ECs) were subjected to experimental ischemia, and conditioned medium (CM) from each was collected and analyzed to assess myogenic and/or angiogenic potential. In muscle progenitors, mRNA expression of VEGF and its cognate receptors (Nrp1, Flt, Flk) was present and decreased during myotube formation in vitro, and EC CM or VEGF increased myoblast proliferation. Angiopoietin-1 (Ang-1), Tie1, and Tie2 mRNA increased during MC differentiation in vitro. Exogenous Ang-1 enhanced myogenic (MyoD and Myogenin) mRNA in differentiating myoblasts and increased myosin heavy chain protein. Myotube formation was enhanced by MC CM and inhibited by EC CM. Ang-1 protein was present in CM from MCs isolated from both the genetically ischemia-susceptible BALB/c and ischemia-resistant C57BL/6 mouse strains, and chimeric Tie2 receptor trapping in situ ablated Ang-1's myogenic effects in vitro. Ang-1 or MC CM enhanced myotube formation in a mixed isolate of muscle progenitors as well as a myoblast co-culture with pluripotent mesenchymal cells (10T1/2) and this effect was abrogated by viral expression of the extracellular domain of Tie2 (AdsTie2). Furthermore, mesh/tube formation by HUVECs was enhanced by Ang-1 or MC CM and abrogated by Tie2 chimeric receptor trapping. Our results demonstrate the ability of muscle and endothelial cell-derived vascular growth factors, particularly Ang-1, to serve as multi-functional stimuli regulating crosstalk between blood vessels and muscle cells during regeneration from ischemic myopathy.
Highlights
Peripheral artery disease (PAD) is caused by atherosclerosis of the peripheral arteries, most commonly in the lower extremities, and is nearly as prevalent as coronary artery disease (CAD) (Hirsch et al, 2006)
The proliferative effect of recombinant vascular endothelial growth factor (VEGF) on muscle progenitor cell (MPC) was replicated with conditioned medium generated from experimentally ischemic endothelial cells (EC CM), but not muscle cells (MC CM) (Figure 1F), indicating the potential for ischemic endothelial cells to drive myoblast proliferation and subsequent muscle regeneration in the ischemic environment
In this report we demonstrate that muscle and endothelial cells exposed to experimental ischemia in vitro secrete angiogenic factors that are capable of regulating muscle progenitor cell proliferation and muscle progenitor and pluripotent cell differentiation into myotubes, as well as endothelial tube formation
Summary
Peripheral artery disease (PAD) is caused by atherosclerosis of the peripheral arteries, most commonly in the lower extremities, and is nearly as prevalent as coronary artery disease (CAD) (Hirsch et al, 2006). Research on the limb tissue response to ischemia has focused on angiogenesis and vascular remodeling. In addition to delivery of exogenous vascular growth factors, therapeutic angiogenesis by targeted delivery of transduced myoblasts has been suggested as a promising option for ischemic muscle diseases (Von Degenfeld et al, 2003), and clinical research has recently emphasized the development of stem or progenitor cell therapies to stimulate angiogenesis in patients with PAD (Prather et al, 2009; Fadini et al, 2010, 2012; Lawall et al, 2011; Blum et al, 2012; Volz et al, 2012). In addition to stimulating vascular growth through actions on endothelial cells (ECs), it has been suggested that these approaches may act directly on muscle progenitor cells (Deasy et al, 2009). The presence of muscle degenerative and regenerative phenotypes in both mouse hindlimb ischemia (HLI) and human PAD are often overlooked or minimized (Yang et al, 2008)
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