Background— Peripheral arterial disease (PAD) caused by occlusive atherosclerosis of the lower extremity has 2 major clinical manifestations. Critical limb ischemia is characterized by rest pain and/or tissue loss and has a ≥40% risk of death and major amputation. Intermittent claudication causes pain on walking, has no tissue loss, and has amputation plus mortality rates of 2% to 4% per year. Progression from claudication to limb ischemia is infrequent. Risk factors in most PAD patients overlap. Thus, we hypothesized that genetic variations may be linked to presence or absence of tissue loss in PAD. Methods and Results— Hindlimb ischemia (murine model of PAD) was induced in C57BL/6, BALB/c, C57BL/6×BALB/c (F1), F1×BALB/c (N2), A/J, and C57BL/6J-Chr7 A/J /NaJ chromosome substitution strains. Mice were monitored for perfusion recovery and tissue necrosis. Genome-wide scanning with polymorphic markers across the 19 murine autosomes was performed on the N2 mice. Greater tissue loss and poorer perfusion recovery occurred in BALB/c than in the C57BL/6 strain. Analysis of 105 N2 progeny identified a single quantitative trait locus on chromosome 7 that exhibited significant linkage to both tissue necrosis and extent of perfusion recovery. Using the appropriate chromosome substitution strain, we demonstrate that C57BL/6-derived chromosome 7 is required for tissue preservation. Conclusions— We have identified a quantitative trait locus on murine chromosome 7 (LSq-1) that is associated with the absence of tissue loss in a preclinical model of PAD and may be useful in identifying gene(s) that influence PAD in humans.

Background Micro-RNAs (miRs) are key regulators of gene expression in response to injury, but there is limited knowledge of their role in ischemia-induced angiogenesis, such as in peripheral arterial disease (PAD). Here, we used an unbiased strategy and took advantage of different phenotypic outcomes that follow surgically induced hind-limb ischemia (HLI) between inbred mouse strains to identify key micro-RNAs involved in perfusion recovery from HLI. Methods and Results From comparative micro-RNA profiling between inbred mouse strains which display profound differences in their extent of perfusion recovery following HLI, we found that the mouse strain with higher levels of miR-93 in hind-limb muscle before ischemia, and the greater ability to up-regulate miR-93 in response to ischemia had better perfusion recovery. In-vitro, over-expression of miR-93 attenuated hypoxia-induced apoptosis in both endothelial and skeletal muscle cells, and enhanced proliferation in both cell types. In addition, miR-93 over-expression enhanced endothelial cell tube formation. In-vivo, miR-93 over-expression enhanced capillary density and perfusion recovery from hind-limb ischemia, and antagomirs to miR-93 attenuated perfusion recovery. Both in-vitro and in-vivo modulation of miR-93 resulted in alterations in the expression of more than one cell cycle pathway gene in two different cell types. Conclusions Our data indicate that miR-93 enhances perfusion recovery from hind-limb ischemia by modulation of multiple genes that coordinate the functional pathways of cell proliferation and apoptosis. Thus, miR-93 is a strong potential target for pharmacological modulation to promote angiogenesis in ischemic tissue.

Abstract-Deficient angiogenesis after ischemia may contribute to worse outcomes of peripheral arterial disease in patients with diabetes mellitus (DM). Vascular endothelial growth factor (VEGF) and its receptors promote angiogenesis. We hypothesized that in peripheral arterial disease, maladaptive changes in VEGF ligand/receptor expression could account for impaired angiogenesis in DM. Skeletal muscle from diet-induced, type 2 diabetic (DM) and age-matched normal chow (NC)-fed mice was collected at baseline and 3 and 10 days after hindlimb ischemia and analyzed for expression of VEGF (nϭ10 per