Peripheral arterial disease (PAD) is a major complication of systemic atherosclerosis which affects approximately 12 million Americans and puts them at risk for lower extremity amputation and death. The presence of diabetes (DM) in individuals with PAD further increases the risk of amputation and death compared to PAD alone. Current medical treatments target systemic atherosclerosis but are not able to improve perfusion to the ischemic limb and directly treat the problem in PAD. There is a pressing clinical need for therapeutic approaches for PAD. Using an established preclinical model of PAD (hind limb ischemia), we previously showed that a quantitative trait locus on the short arm of mouse chromosome 7 (termed LSQ-1) determined perfusion recovery outcomes. Here we have further refined this locus to a region containing 25 genes. Through gene expression profiling we identified 5 genes that are differentially expressed in the ischemic hind limbs of the 2 mouse strains (C57BL/6 and Balb/c) that were used in the original QTL identification. Additionally, we identified ADAM12 as the most differentially expressed of the 5 genes and explored its role in perfusion recovery. We find that ADAM12 is : a) up-regulated in ischemic mouse hind limbs in C57BL/6 mice and in endothelial cells (ECs) exposed to simulated ischemia, b) a knock-down of ADAM12 in vivo in C57BL/6 mice was sufficient to impair perfusion recovery and in vitro decreased EC proliferation, tube formation and survival in simulated ischemia, c) over-expression of ADAM12 in vivo in the Balb/c mouse strain that had poor up-regulation of ADAM12 was sufficient to improve its perfusion recovery following HLI and in vitro increased EC proliferation and survival in hypoxia. These effects of ADAM12 were at least in part, through activation of the receptor tyrosine kinase Tie2. Additionally, we find that in humans with PAD a single nucleotide polymorphism (SNP) in ADAM12 is associated with critical limb ischemia, a severe form of PAD. This data suggests that ADAM12 may play a central role in PAD outcomes. To better understand ADAM12 regulation in ischemia we explored the role of miR29a, a microRNA shown to suppress ADAM12 expression in fibroblast. We find that miR29a overexpression can suppress endothelial cell ADAM12 up-regulation in ischemia. Additionally, we and others have shown that the same C57BL/6 strain that upregulates ADAM12 robustly and recovers favorable following HLI, recovers poorly when DM1 or DM2 is superimposed. Therefore we analyzed miR29a and ADAM12 expression in ischemic hind limbs of C57BL/6 mice with DM1 or DM2 compared to DM C57BL/6 without DM. We found in C57BL/6 mice with DM1 or DM2 miR29a expression is not suppressed and the expression of ADAM12 is blunted. Consistent with these preclinical findings, we also found human skeletal muscle biopsy from PAD patients with DM show elevated levels of miR29a compared to those with PAD alone. Taken together this results we form the central hypothesis that in the absence of DM, PAD/HLI suppresses miR29a expression, resulting in up-regulation of ADAM12. Dysregulation of this pathway in DM contributes to poorer PAD outcomes.
Aim 1 Determine whether miR29a negatively regulates ADAM12 expression in the ischemic hind limbs and whether this is sufficient to impair perfusion recovery and increase tissue loss following HLI.
Aim 2 will investigate the therapeutic potential of systemic delivery of anti-miR29a compared to systemic delivery of ADAM12 gene on perfusion recovery and tissue loss in mice with type 1 or type 2 DM following HLI. We will establish the effects of these therapeutic strategies on arteriogenesis in the abductor muscles, angiogenesis and circulating progenitors in the ischemic hind limbs.
Aim 3 will use an existing human skeletal muscle bio-bank to determine whether a) miR29a and ADAM12 protein expression differs in skeletal muscles of individuals with PAD versus controls without PAD. b) In individuals with PAD we will establish whether miR29a and ADAM12 protein expression in the lower extremity skeletal muscles differ dependent on DM status. c) Across all subjects with PAD, determine whether lower extremity skeletal muscle miR29a and ADAM12 expression will correlate with clinical measures of PAD severity (ankle brachial index and peak walking time).

Public Health Relevance

Peripheral arterial disease (PAD) of the lower extremities is the result of arteriosclerotic blockage of blood vessels and its severity varies even among people with similar occlusions, suggesting a possible role for genetics in its severity. Individual with PAD and diabetes are at much higher risk of amputation and death. We have identified a gene called ADAM12 that improves recovery of blood flow after a vessel is blocked. We have found that diabetes appears to impair regulation of this gene in humans and in rodents such that its expression is blunted in muscles with poor blood flow. Therefore we would like to better understand how this gene works to improve blood flow and determine how to manipulate this for therapy in PAD especially in individuals with diabetes.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
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Vascular Cell and Molecular Biology Study Section (VCMB)
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Galis, Zorina S
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University of Tennessee Health Science Center
Internal Medicine/Medicine
Schools of Medicine
United States
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Okeke, Emmanuel; Dokun, Ayotunde O (2018) Role of genetics in peripheral arterial disease outcomes; significance of limb-salvage quantitative locus-1 genes. Exp Biol Med (Maywood) 243:190-197
Chen, Lingdan; Okeke, Emmanuel; Ayalew, Dawit et al. (2017) Modulation of miR29a improves impaired post-ischemic angiogenesis in hyperglycemia. Exp Biol Med (Maywood) 242:1432-1443
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