Modulation of miR29a and ADAM12 to improve peripheral arterial disease outcomes in diabetes

调节 miR29a 和 ADAM12 可改善糖尿病外周动脉疾病的预后

• 批准号: 9008921
• 负责人: Ayotunde Oluropo Dokun
• 金额: $ 39.5万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-05-01 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9008921
• 关键词: Affect; American; Amputation; Ankle; Atherosclerosis; Biological Assay; Biopsy; Blood Vessels; Blood flow; C57BL/6 Mouse; Cell Proliferation; Cessation of life; Chromosomes, Human, Pair 7; Clinical; Complication; Data; Diabetes Mellitus; Disease Outcome; Endothelial Cells; Fibroblasts; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic; Genetic Polymorphism; Human; Hypoxia; Immunoprecipitation; In Vitro; Individual; Ischemia; Limb structure; Lower Extremity; Measures; Mediating; Medical; Messenger RNA; MicroRNAs; Mouse Strains; Mus; Muscle; Necrosis; Non-Insulin-Dependent Diabetes Mellitus; Outcome; Pathway interactions; Patients; Perfusion; Peripheral arterial disease; Play; Pre-Clinical Model; Quantitative Trait Loci; RNA; Receptor Protein-Tyrosine Kinases; Recovery; Regulation; Risk; Rodent; Role; Severities; Severity of illness; Single Nucleotide Polymorphism; Skeletal Muscle; Testing; Therapeutic; Therapeutic Effect; Time; Tissues; Tube; Up-Regulation; Walking; Work; angiogenesis; arm; base; differential expression; high risk; improved; in vivo; indexing; knock-down; overexpression; pre-clinical; prevent; progenitor; protein expression; public health relevance

 DESCRIPTION (provided by applicant):¨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).

 描述（由申请人提供）：？外周动脉疾病（PAD）是全身动脉粥样硬化的主要并发症，影响约1200万美国人，并使他们处于下肢截肢和死亡的风险中。与单独的PAD相比，PAD患者中糖尿病（DM）的存在进一步增加了截肢和死亡的风险。目前的医学治疗靶向全身动脉粥样硬化，但不能改善缺血肢体的灌注并直接治疗PAD中的问题。临床上迫切需要PAD的治疗方法。 使用已建立的PAD（后肢缺血）临床前模型，我们先前表明，小鼠7号染色体短臂上的数量性状位点（称为LSQ-1）决定灌注恢复结果。在这里，我们进一步将该基因座细化到包含25个基因的区域。通过基因表达谱分析，我们确定了5个基因，在缺血后肢的2个小鼠品系（C57 BL/6和Balb/c），用于原始QTL鉴定的差异表达。此外，我们确定了ADAM 12作为5个基因中表达差异最大的基因，并探讨了其在灌注恢复中的作用。我们发现ADAM 12是：a）在C57 BL/6小鼠的缺血小鼠后肢中和暴露于模拟缺血的内皮细胞（EC）中上调，B）在C57 BL/6小鼠中体内敲低ADAM 12足以损害灌注恢复，并且在模拟缺血中体外降低EC增殖、管形成和存活，c）在ADAM 12上调较差的Balb/c小鼠品系中，体内过表达ADAM 12足以改善其在HLI后的灌注恢复，并且体外增加EC增殖和缺氧中的存活。ADAM 12的这些作用至少部分是通过激活受体酪氨酸激酶Tie 2实现的。此外，我们发现在PAD患者中，ADAM 12的单核苷酸多态性（SNP）与严重的肢体缺血（PAD的一种严重形式）相关。这些数据表明ADAM 12可能在PAD结果中发挥核心作用。 为了更好地理解缺血中的ADAM 12调节，我们探索了miR 29 a的作用，miR 29 a是一种显示抑制成纤维细胞中ADAM 12表达的microRNA。我们发现miR 29 a过表达可以抑制缺血时内皮细胞ADAM 12的上调。此外，我们和其他人已经表明，同样的C57 BL/6菌株，上调ADAM 12强劲，并恢复良好后HLI，恢复差时，DM 1或DM 2叠加。因此，我们分析了与无DM的DM C57 BL/6小鼠相比，DM 1或DM 2的C57 BL/6小鼠缺血后肢中的miR 29 a和ADAM 12表达。我们发现在C57 BL/6小鼠中，DM 1或DM 2的miR 29 a表达未被抑制，而ADAM 12的表达被钝化。与这些临床前发现一致，我们还发现来自患有DM的PAD患者的人骨骼肌活检显示与仅患有PAD的患者相比miR 29 a水平升高。 综合这些结果，我们形成了核心假设，即在不存在DM的情况下，PAD/HLI抑制miR 29 a表达，导致ADAM 12上调。糖尿病患者中该通路的失调导致PAD结局较差。目的1确定miR 29 a是否负调节缺血后肢中的ADAM 12表达，以及这是否足以损害灌注恢复并增加HLI后的组织损失。目的2将研究与全身递送ADAM 12基因相比，全身递送抗miR 29 a对HLI后1型或2型DM小鼠的灌注恢复和组织损失的治疗潜力。我们将建立这些治疗策略对展肌动脉生成、缺血后肢血管生成和循环祖细胞的影响。目的3将使用现有的人类骨骼肌生物库来确定a）在患有PAD的个体与没有PAD的对照的骨骼肌中miR 29 a和ADAM 12蛋白表达是否不同。B）在患有PAD的个体中，我们将确定下肢骨骼肌中的miR 29 a和ADAM 12蛋白表达是否取决于DM状态而不同。c）在所有患有PAD的受试者中，确定下肢骨骼肌miR 29 a和ADAM 12表达是否与PAD严重程度的临床测量（踝臂指数和峰值步行时间）相关。