Fig 1: microRNA‐29a (iR‐29a) overexpression did not affect skeletal muscle regeneration, myofiber cross‐sectional area (CSA), but decreased skeletal muscle function in experimental peripheral artery disease.Day 14 postischemic tibialis anterior (TA) sections treated with empty vector (EV) or miR‐29a were subjected to analysis. A and B, Representative images of eMyHC‐stained sections showing regenerating muscle fibers (Ischemia+EV: n=4 and Ischemia+miR‐29a: n=5, scale bar=20 μm). C, eMyHC+myofiber as an index of myofiber regeneration (Ischemia+EV: n=4 and Ischemia+miR‐29a: n=4, P=0.62). D, Mean myofiber CSA (Ischemia+EV: n=4 and Ischemia+miR‐29a: n=4, P=0.80). E, Maximal isometric torque of ankle dorsiflexors of the TA at day 14 following ischemia (Ischemia+EV: n=5 and Ischemia+miR‐29a: n=4, *P<0.05).
Fig 2: Treatment of ischemic mouse hind limbs with microRNA‐29a (miR‐29a) impairs ischemia‐induced ADAM12 (a disintegrin and metalloproteinase gene 12) upregulation. A, Higher miR‐29a is expressed in the ischemic gastrocnemius of mice treated with miR‐29a compared with the Ischemia+Empty Vector (EV) group (Ischemia+EV: n=5 and Ischemia+miR‐29a: n=9, **P<0.005). B, Lower ADAM12 messenger RNA is expressed in the ischemic gastrocnemius of mice treated with miR‐29a compared with controls (Ischemia+miR‐29aEV: n=5 and Ischemia+miR‐29a: n=9, **P<0.005). C, Western blotting showing lower ADAM12 protein expression in gastrocnemius lysates from mouse hindlimbs that were treated with miR‐29a compared with controls. D, quantification of Western blot bands (Ischemia+EV: n=5 and Ischemia+miR‐29a: n=5, **P<0.005). All analysis were performed using samples from day 3 postischemic gastrocnemius muscles. GAPDH, indicates glyceraldehyde‐3‐phosphate dehydrogenase.
Fig 3: Overexpression of microRNA‐29a (miR‐29a) in ischemic mouse hind limbs impairs postischemic perfusion recovery. A, Representative laser speckle contrast imaging illustrating decreased blood perfusion in postischemic hindlimbs of mice treated with miR‐29a. B, Quantification of perfusion recovery following hind limb ischemia (HLI) at various time points. Vertical axis shows extent of perfusion in the ischemic limb relative to the nonischemic limb. Horizontal axis shows days following HLI (day 14, Ischemia+Control: n=5, Ischemia+EV: n=7 and Ischemia+miR‐29a: n=8, *P<0.05). EV indicates empty vector.
Fig 4: RNA immunoprecipitation–based anti‐miR competitive assay reveals dose‐dependent displacement of ADAM12 (a disintegrin and metalloproteinase gene 12) messenger RNA (mRNA) from the AGO‐2 (Argonaut‐2) complex in mouse skeletal microvascular endothelial cells (msMVEC).msMVEC cell lysates were treated with different concentrations of anti‐miRs (0, 4, 40, 400 nmol/L) followed by immunoprecipitation with AGO‐2 antibody, and the microRNA and mRNA associated with the AGO‐2 complex was analyzed by real time–quantitative polymerase chain reaction. microRNA levels were normalized to miR‐10a. 8 A through C, microRNA‐29a (miR‐29a), ADAM12, and ADAM17 (a disintegrin and metalloproteinase gene 17) mRNA levels in anti‐miR‐29a–treated lysates. (D through F) miR‐29a, ADAM12, and ADAM17 mRNA levels in control anti‐miR‐696–treated lysates (n=3–4 per group, **P<0.005 and *P<0.05). UT indicates untreated; HPRT, hypoxanthine‐guanine phosphoribosyltransferase.
Fig 5: microRNA‐29a (miR‐29a) overexpression increased the extent of skeletal muscle injury in experimental peripheral artery disease.Day 14 postischemic tibialis anterior from treatment control as well as sections treated with control, empty vector (EV), or miR‐29a were subjected to analysis. A through C, Representative images of hematoxylin and eosin–stained sections showing centrally located nuclei in muscle fibers. D, Quantification of skeletal muscle injury as determined by the percentage of total number of muscle fibers with centrally located nuclei (Ischemia+Control: n=5, Ischemia+EV: n=4 and Ischemia+miR‐29a: n=3, *P<0.05, scale bar=20 μm).
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