Division of Endocrinology and Metabolism, Carver College of Medicine University of Iowa Iowa City IA.
Fraternal Order of Eagles Diabetes Research Center, Carver College of Medicine University of Iowa Iowa City IA.
J Am Heart Assoc. 2022 Aug 16;11(16):e025727. doi: 10.1161/JAHA.122.025727. Epub 2022 Aug 10.
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.
外周动脉疾病是由心脏以外的血管发生动脉粥样硬化阻塞引起的，最常影响下肢血管。血管生成是参与外周动脉疾病恢复血流的缺血后适应的一部分。以前，在小鼠后肢缺血性外周动脉疾病模型中，我们将 ADAM12（解整合素和金属蛋白酶基因 12）鉴定为缺血后灌注恢复的关键遗传修饰因子。然而，关于缺血时 ADAM12 的调节知之甚少。微小 RNA 是一类小的、非编码的、单链 RNA，主要通过信使 RNA（mRNA）的转录抑制来调节基因表达。我们显示微小 RNA-29a（miR-29a）在糖尿病和缺血的情况下调节 ADAM12 的表达。然而，miR-29a 如何调节 ADAM12 尚不清楚。此外，miR-29a 调节在非糖尿病环境中的生理影响尚不清楚。方法和结果：我们在缺血的小鼠腓肠肌和胫骨前肌中过表达或抑制 miR-29a，并定量分析其对灌注恢复、ADAM12 表达、血管生成和骨骼肌再生的影响。此外，我们使用基于 RNA 免疫沉淀的抗-miR 竞争测定法，研究了 miR-29a 和 ADAM12 mRNA 在小鼠微血管内皮细胞、骨骼肌和人内皮细胞裂解物中的相互作用。在缺血性小鼠后肢中异位表达 miR-29a 会降低 ADAM12 mRNA 表达，增加骨骼肌损伤，降低骨骼肌功能，并减少血管生成和灌注恢复，但对后肢缺血后的骨骼肌再生和肌纤维横截面积没有影响。基于 RNA 免疫沉淀的抗-miR 竞争测定研究表明，miR-29a 拮抗剂以剂量依赖性方式从 AGO-2（Argonaut-2）复合物中置换 miR-29a 和 ADAM12 mRNA。结论：综上所述，数据表明 miR-29a 通过直接与其 mRNA 结合来抑制 ADAM12 的表达，导致骨骼肌功能、血管生成和灌注不良受损。因此，糖尿病和衰老时 miR-29a 水平升高可能导致血管病变，而 miR-29a 的调节可能成为一种治疗靶点。
