Both Type 1 diabetes mellitus (DM1) and type 2 diabetes mellitus (DM2) are associated with an increased risk of limb amputation in peripheral arterial disease (PAD). How diabetes contributes to poor PAD outcomes is poorly understood but may occur through different mechanisms in DM1 and DM2. Previously, we identified a disintegrin and metalloproteinase gene 12 (ADAM12) as a key genetic modifier of post-ischemic perfusion recovery. In an experimental PAD, we showed that ADAM12 is regulated by miR-29a and this regulation is impaired in ischemic endothelial cells in DM1, contributing to poor perfusion recovery. Here we investigated whether miR-29a regulation of ADAM12 is altered in experimental PAD in the setting of DM2. We also explored whether modulation of miR-29a and ADAM12 expression can improve perfusion recovery and limb function in mice with DM2. Our result showed that in the ischemic limb of mice with DM2, miR-29a expression is poorly downregulated and ADAM12 upregulation is impaired. Inhibition of miR-29a and overexpression of ADAM12 improved perfusion recovery, reduced skeletal muscle injury, improved muscle function, and increased cleaved Tie 2 and AKT phosphorylation. Thus, inhibition of miR-29a and or augmentation of ADAM12 improves experimental PAD outcomes in DM2 likely through modulation of Tie 2 and AKT signalling.
B-cell lymphoma 2 (Bcl-2)-associated athanogene 3 (BAG3) protein is a member of BAG family of co-chaperones that modulates major biological processes, including apoptosis, autophagy, and development to promote cellular adaptive responses to stress stimuli. Although BAG3 is constitutively expressed in several cell types, its expression is also inducible and is regulated by microRNAs (miRNAs). miRNAs are small non-coding RNAs that mostly bind to the 3′-UTR (untranslated region) of mRNAs to inhibit their translation or to promote their degradation. miRNAs can potentially regulate over 50% of the protein-coding genes in a cell and therefore are involved in the regulation of all major functions, including cell differentiation, growth, proliferation, apoptosis, and autophagy. Dysregulation of miRNA expression is associated with pathogenesis of numerous diseases, including peripheral artery disease (PAD). BAG3 plays a critical role in regulating the response of skeletal muscle cells to ischemia by its ability to regulate autophagy. However, the biological role of miRNAs in the regulation of BAG3 in biological processes has only been elucidated recently. In this review, we discuss how miRNA may play a key role in regulating BAG3 expression under normal and pathological conditions.
Background Peripheral artery disease is caused by atherosclerotic occlusion of vessels outside the heart and most commonly affects vessels of the lower extremities. Angiogenesis is a part of the postischemic adaptation involved in restoring blood flow in peripheral artery disease. Previously, in a murine hind limb ischemia model of peripheral artery disease, we identified ADAM12 (a disintegrin and metalloproteinase gene 12) as a key genetic modifier of postischemic perfusion recovery. However, less is known about ADAM12 regulation in ischemia. MicroRNAs are a class of small, noncoding, single‐stranded RNAs that regulate gene expression primarily through transcriptional repression of messenger RNA (mRNA). We showed microRNA‐29a (miR‐29a) modulates ADAM12 expression in the setting of diabetes and ischemia. However, how miR‐29a modulates ADAM12 is not known. Moreover, the physiological effects of miR‐29a modulation in a nondiabetic setting is not known. Methods and Results We overexpressed or inhibited miR‐29a in ischemic mouse gastrocnemius and tibialis anterior muscles, and quantified the effect on perfusion recovery, ADAM12 expression, angiogenesis, and skeletal muscle regeneration. In addition, using RNA immunoprecipitation–based anti‐miR competitive assay, we investigated the interaction of miR‐29a and ADAM12 mRNA in mouse microvascular endothelial cell, skeletal muscle, and human endothelial cell lysates. Ectopic expression of miR‐29a in ischemic mouse hind limbs decreased ADAM12 mRNA expression, increased skeletal muscle injury, decreased skeletal muscle function, and decreased angiogenesis and perfusion recovery, with no effect on skeletal muscle regeneration and myofiber cross‐sectional area following hind limb ischemia. RNA immunoprecipitation–based anti‐miR competitive assay studies showed miR‐29a antagomir displaced miR‐29a and ADAM12 mRNA from the AGO‐2 (Argonaut‐2) complex in a dose dependent manner. Conclusions Taken together, the data show miR‐29a suppresses ADAM12 expression by directly binding to its mRNA, resulting in impaired skeletal muscle function, angiogenesis, and poor perfusion. Hence, elevated levels of miR‐29a, as seen in diabetes and aging, likely contribute to vascular pathology, and modulation of miR‐29a could be a therapeutic target.
Diabetes Mellitus (DM) is a major risk factor for developing peripheral arterial disease (PAD) and individuals with DM have worse PAD outcomes but the molecular mechanisms involved are poorly understood. Previously, in a hind limb ischemia (HLI) model of PAD, we identified a disintegrin and metalloproteinase gene 12 (ADAM12) as a key genetic modifier of post-ischemic perfusion recovery. Moreover, we showed that expression of ADAM12 in mouse and human tissue is regulated by miR29a. In non-diabetic mice, miR29a expression is downregulated after HLI that allows increased expression of ADAM12. However, upon HLI in high fat diet feed (HFD) mice, a model of type 2 diabetes, miR29a expression remains elevated that prevents ADAM12 increase and results in poor reperfusion recovery, increased skeletal muscle injury and decreased muscle function. Hence, we hypothesized that inhibition of miR29a or augmenting ADAM12 would improve these functional outcomes. Mice (male, 26–28 weeks old) were randomized into 3 treatment groups and their hind limbs were treated with saline (grp1), ADAM12 cDNA (grp 2) or mir29a-inhibitor (grp3), through targeted micro-bubble delivery. Mice were treated at -3 days and -1 pre-surgery, followed by post-surgery weekly boosting. HLI was achieved by unilateral ligation and excision of the femoral artery of the left hind limb. The right hind limb served as non-ischemic control. Gene expression analysis in the hind limbs 3 days post HLI showed decreased miR29a expression in normal chow fed B6, but elevated miR29a expression in HFD (B6 vs HFD; 0.5730±0.01 vs.1.02 ± 0.06, n=3–4, p= 0.001). Treatment with miR29a inhibitor decreased miR29a expression in HFD and increased ADAM12 expression compared to control untreated HFD mice (miR29a INH vs Control HFD: 0.70±0.06 vs 1.02±0.06, n= 4–5, p= 0.004) ADAM12 expression (miR29A INH vs Control: HFD 208.62±24.52 vs 11.75±4.94, n= 3–4 P<0.01). Although ADAM12 cDNA improved ADAM12 expression, miR29a inhibition increased ADAM12 expression to a greater extent (HFD vs ADAM12 vs miR29aINH; 11.75±4.94 vs 20.71±2.98 vs 208.62±24.52, n3-4, p=< 0.001). Accordingly, miR29a inhibition and ADAM12 augmentation decreased skeletal muscle injury assessed by the number of centralized nuclei/muscle fibre (Control vs ADAM12 vs miR29aINH: 0.252±0.043, vs 0.139±0.041 vs 0.040±0.012 n=4, p= 0.05), and improved skeletal muscle function assessed as maximum muscle contraction (Control vs ADAM12 vs miR29aINH: 0.17±0.06 vs 0.26±0.06, vs 0.54±0.08, n=6–7, p<0.01). It also improved perfusion recovery, (% ischemic to non-ischemic limb, control vs ADAM12 vs miR29aINH: 42.52±5.35, vs 58.45±4.87, vs 97.59±6.14, n= 5–10, p<0.01). Thus, our results show augmentation of ADAM12 and Inhibition of MiR29a improves outcomes in experimental PAD in diabetic mice but inhibiting miR29a is a more effective strategy. 2414 characters now2500 characters allowed
Peripheral artery disease (PAD) is caused by atherosclerotic occlusion of vessels outside the heart and most commonly affects vessels of the lower extremities. Angiogenesis is a part of the post ischemic adaption involved in restoring blood flow in PAD. Previously, in a murine hind limb ischemia (HLI) model of PAD, we identified ADAM12 as a key genetic modifier of post-ischemic perfusion recovery. However, less is known about ADAM12 regulation in ischemia. MiRNAs are a class of small, non-coding, single-stranded RNAs that regulate gene expression primarily through transcriptional repression of mRNA. We showed miR29a modulates ADAM12 expression in the setting of type 1 DM and ischemia. However, how miR29a modulates ADAM12 was not known. Moreover, the physiological effects of miR29a modulation in a non-diabetic setting was not known. Here, we demonstrate that AAV-mediated ectopic overexpression of miR29a in ischemic mouse hind limbs impairs post ischemic perfusion recovery and angiogenesis. We further demonstrate that miR29a regulates ADAM12 through direct interaction with ADAM12 mRNA. Treatment of ischemic mouse hind limbs with AAV9 particles containing miR29a (pAV-miR29a) increased miR29a expression (pAV-control vs pAV-miR29a: 3.22 ± 0.36, vs 1.0 ± 0.09, p<0.05, n=5-6,) and decreased ADAM12 mRNA expression (1.0 ± 0.31 vs 0.30 ± 0.08, p<0.05, n=7-10) resulting in decreased angiogenesis (1.0 ± 0.07 vs 0.66 ± 0.04, p<0.05, n=5), and impairment perfusion (week 3 post HLI perfusion, 101.9±5.55 vs 66.13±12.39, p<0.05, n=5-6,).AGO2 miRNA/RNA-immunoprecipitation studies showed levels of miR29a and ADAM12 mRNA in the complex decreased in samples treated with antagomiR29a (4, 40, 400nM) in a dose dependent manner (miR29a: 0.87, 0.68, 0.44, and ADAM12: 0.89, 0.78, 0.67, fold) but was unchanged by control antagomir (miR29a: 0.84, 0.84, 0.87, fold, and ADAM12: 1.11, 1.12, 1.10, fold). Taken together the data shows miR29a suppresses ADAM12 expression by directly binding to its mRNA resulting in impaired angiogenesis and poor perfusion. Hence, elevated levels of miR29a as seen in diabetes likely contributes to vascular pathology and lowering miR29a could be a therapeutic target.
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