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

 描述(申请人提供)：？外周动脉疾病(PAD)是系统性动脉粥样硬化的主要并发症，影响着大约1200万美国人，使他们面临截肢和死亡的风险。与单纯PAD相比，PAD患者中存在糖尿病(DM)进一步增加了截肢和死亡的风险。目前的医学治疗针对的是系统性动脉粥样硬化，但不能改善缺血肢体的血流灌注，也无法直接治疗PAD问题。临床上迫切需要PAD的治疗方法。利用已建立的PAD(后肢缺血)临床前模型，我们先前证明了小鼠7号染色体短臂上的一个数量性状基因座(称为LSQ-1)决定了血流灌注恢复的结果。在这里，我们将该基因座进一步细化到一个包含25个基因的区域。通过基因表达谱分析，我们找到了用于原始QTL定位的两个品系(C57BL/6和Balb/c)在缺血后肢中差异表达的5个基因。此外，我们确定ADAM12是这5个基因中表达差异最大的基因，并探讨了它在血流恢复中的作用。我们发现：a)ADAM12在C57BL/6小鼠缺血后肢和暴露于模拟缺血的内皮细胞(ECs)中上调，b)ADAM12在C57BL/6小鼠体内的表达下调足以损害灌注恢复，体外降低模拟缺血中的EC增殖、管子形成和存活，c)ADAM12在体内的高表达足以促进HLI后的灌注恢复，体外可增加EC的增殖和缺氧下的存活。ADAM12的这些作用至少部分是通过激活受体酪氨酸激酶Tie2来实现的。此外，我们发现在患有PAD的人中，ADAM12的单核苷酸多态(SNP)与严重的肢体缺血有关，这是PAD的一种严重形式。这一数据表明ADAM12可能在PAD的转归中起核心作用。为了更好地了解ADAM12在缺血中的调控，我们探索了miR29a的作用，miR29a是一种被证明抑制成纤维细胞中ADAM12表达的微RNA。我们发现miR29a过表达可以抑制缺血时内皮细胞ADAM12的上调。此外，我们和其他人已经证明，同一株C57BL/6菌株在HLI后强劲上调ADAM12并恢复良好，但当DM1或DM2重叠时恢复较差。因此，我们分析了使用DM1或DM2的C57BL/6小鼠缺血后肢中miR29a和ADAM12的表达，并与未使用DM的C57BL/6小鼠进行了比较。我们发现，在带有DM1或DM2的C57BL/6小鼠中，miR29a的表达没有受到抑制，ADAM12的表达被钝化。与这些临床前发现一致的是，我们还发现，与单纯PAD患者相比，PAD合并DM患者的骨骼肌活检显示miR29a水平升高。综合这些结果，我们形成了一个中心假设，即在没有DM的情况下，PAD/HLI抑制miR29a的表达，导致ADAM12的上调。糖尿病患者这一途径的失调会导致较差的PAD结局。目的1确定miR29a是否负性调节缺血后肢ADAM12的表达，以及这是否足以损害HLI后的灌流恢复和增加组织丢失。目的比较系统注射抗miR29a抗体和系统注射ADAM12基因对1型或2型糖尿病小鼠HLI后血流灌注恢复和组织丢失的治疗作用。我们将确定这些治疗策略对外展肌的动脉生成、缺血后肢的血管生成和循环祖细胞的影响。AIM 3将使用现有的人类骨骼肌生物库来确定a)miR29a和ADAM12蛋白在患有PAD的个体和没有PAD的对照组的骨骼肌中的表达是否存在差异。B)在PAD患者中，我们将确定miR29a和ADAM12蛋白在下肢骨骼肌中的表达是否与DM状态有关。C)在所有患有PAD的受试者中，确定下肢骨骼肌miR29a和ADAM12的表达是否与PAD严重程度的临床测量(踝臂指数和高峰步行时间)相关。

 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).