Peripheral artery disease (PAD) is a major cause of human morbidity and mortality. Evidence suggests that the most severe manifestation of PAD, critical limb ischemia (CLI), is clinically distinct from the more benign syndrome of intermittent claudication (IC). In mice, the propensity to develop CLI-like tissue necrosis is strain- dependent. Both susceptible (BALB/c) and resistant (C57BL/6) strains have been identified, suggesting that a similar genetic susceptibility exists in humans. Human genetic studies have demonstrated linkages to PAD, however the mechanisms that predispose to CLI vs. IC remain unknown. One reason for this may be that the vast majority of studies examining susceptibility to tissue necrosis in limb ischemia have focused on the vasculature. However, we have found that the skeletal muscle cell response, particularly that of skeletal muscle progenitor cells (MPCs), is a key determinant of tissue necrosis after limb ischemia in mice and susceptibility to CLI in humans. Moreover, our findings provide a novel mechanistic model that accounts for the role of known modulators of PAD, such as VEGF, in the development of CLI. In preliminary studies, we have developed a murine model of subacute limb ischemia that leads to muscle necrosis similar to that seen in humans with CLI, and we have demonstrated that: 1) in this model, but not in acute ischemia, mice develop large, mature neovessels in the non-ischemic limb;2) these vessels contain cells that co-express the MPC marker Pax7 together with CD31, suggesting that MPCs can differentiate into endothelial cells (ECs);3) ablation of Pax7+ MPCs results in dramatic tissue necrosis, even in necrosis-resistant mouse strains;4) expression of the VEGF receptor VEGFR-2 on MPCs is induced by ischemia in necrosis-resistant but not necrosis-susceptible mice;5) VEGF induces MPC proliferation and differentiation;and 6) loss of VEGFR-2 in MPCs in vivo results in deficient muscle regeneration. Taken together, these findings suggest a model in which MPCs, in response to VEGF stimulation, incorporate into new blood vessels to support tissue perfusion and protect muscle cells from ischemic injury. In addition, VEGF promotes MPCs'known direct contribution to muscle regeneration. Thus, we hypothesize that VEGF receptor signaling in endogenous muscle progenitor cells mediates both skeletal muscle neovascularization and myofiber regeneration after limb ischemia in order to limit muscle necrosis. To investigate this hypothesis, our Specific Aims are to: 1. Determine if MPC VEGF receptors are required for neovascularization and muscle regeneration in vivo. 2. Determine if paracrine VEGF signaling is required for MPC-mediated neovascularization in vivo. 3. Determine if MPC VEGF receptors are required for ischemic MPC proliferation, survival, and differentiation in vitro, and if MPCs are similarly affected in patients with CLI.

Public Health Relevance

Critical limb ischemia (CLI) is a severe manifestation of peripheral artery disease (PAD) that often leads to limb amputation and, as a result, causes significant suffering and an increased risk of death. Recent research indicates that a population of muscle progenitor cells (MPCs) may contribute to both new blood vessel growth and muscle regeneration in the limbs of individuals with PAD. This proposal will investigate the role of the vascular endothelial growth factor (VEGF) receptors in the function of MPCs in the setting of decreased blood supply, such as that which occurs in PAD. Understanding the mechanisms by which these receptors regulate MPC function in PAD may lead to new strategies to prevent tissue loss and subsequent limb amputation in patients with CLI.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
High Priority, Short Term Project Award (R56)
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Vascular Cell and Molecular Biology Study Section (VCMB)
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Reid, Diane M
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Duke University
Internal Medicine/Medicine
Schools of Medicine
United States
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McClung, Joseph M; McCord, Timothy J; Ryan, Terence E et al. (2017) BAG3 (Bcl-2-Associated Athanogene-3) Coding Variant in Mice Determines Susceptibility to Ischemic Limb Muscle Myopathy by Directing Autophagy. Circulation 136:281-296
Padgett, Michael E; McCord, Timothy J; McClung, Joseph M et al. (2016) Methods for Acute and Subacute Murine Hindlimb Ischemia. J Vis Exp :
McClung, Joseph M; McCord, Timothy J; Southerland, Kevin et al. (2016) Subacute limb ischemia induces skeletal muscle injury in genetically susceptible mice independent of vascular density. J Vasc Surg 64:1101-1111.e2
McClung, Joseph M; Reinardy, Jessica L; Mueller, Sarah B et al. (2015) Muscle cell derived angiopoietin-1 contributes to both myogenesis and angiogenesis in the ischemic environment. Front Physiol 6:161
Reinardy, Jessica L; Corey, Daniel M; Golzio, Christelle et al. (2015) Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway. PLoS One 10:e0139614