MicroRNA 93 in Peripheral Arterial Disease

描述（由申请人提供）：外周动脉疾病（PAD）是系统性动脉粥样硬化的主要并发症，影响约1200万美国人。下肢是PAD最常见的部位，目前的医学治疗针对全身性动脉粥样硬化，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。 尽管在不到15年前才被完全认识为真正的生物实体，但现在认识到微RNA（miR）是基因表达的关键调节因子;特别是在对损伤的适应性反应中。miR调节基因表达的最佳表征方法是通过其结合靶mRNA的3'非翻译区并由此降低mRNA表达或蛋白质翻译的能力。然而，单个miR可以通过影响几个功能相关的基因来调节整个生物学途径，从而使miR成为有吸引力的治疗剂。 我们发现miR-93似乎在实验性PAD中起关键作用。HLI后，C57 BL/6（B6）小鼠具有极好的灌注恢复和很少的组织损失。相比之下，在Balb/C小鼠中，灌注恢复是有限的，并且坏死是常见的。利用实验和计算方法，我们确定了mir- 93作为基于应变和缺血的组合而差异表达的最多的miR。miR- 93<0.0001 and >在B6小鼠中而不是在Bab/C中在HLI后显著增加（p增加4倍）。在Balb/C小鼠中，miR-93前体的局部递送导致更大的灌注恢复，而miR-93异构体的全身递送损害了C57/B16小鼠中的灌注恢复。在体外，miR-93的异构体增加了缺氧诱导的细胞凋亡的程度，而用pre-miR-93处理降低了两种细胞类型中缺氧诱导的细胞凋亡的程度。miR-93的拮抗剂减少增殖，而前体miR 93的施用增加增殖。我们一致发现，周期基因p53，E2 F1和p21被改变。总体而言，中心假设miR-93可以作为PAD中出生后血管生成的有效调节剂，尽管其能够单独或与其他潜在靶点一起调节周期基因。具体目标1）：确定 通过在适当的预选时间点检查小鼠，使用功能获得和功能丧失方法，miR-93在体内调节HLI后血流恢复程度中发挥的作用。具体目标2）：利用体外研究确定miR-93诱导的基因表达变化与miR-93/mRNA相互作用直接相关的程度，并确定miR-93诱导的基因表达变化具有细胞和条件特异性的程度。具体目标3）：测量来自具有不同严重程度的PAD的患者以及一组非PAD对照的人血浆和骨骼人样品的已建立库中的miR-93。

DESCRIPTION (provided by applicant): Peripheral arterial disease (PAD) is a major complication of systemic atherosclerosis which affects approximately 12 million Americans. The lower extremity is the most common site for PAD where 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. Though only fully recognized as a true biologic entity less than 15 years ago, micro-RNAs (miRs) which are 15 - 23 nucleotide long RNA's, are now appreciated to be key regulators of gene expression; especially in the adaptive response to injury. The best characterized method by which miRs regulate gene expression is by their ability to bind to the 3' un-translated region of a target mRNA and thereby reducing mRNA expression or protein translation. However, a single miR can regulate entire biologic pathways by affecting several functionally related genes thus making miRs attractive therapeutic agents. We found that miR-93 appears to play a critical role in experimental PAD. Following HLI, C57BL/6 (B6) mice have excellent perfusion recovery and little tissue loss. In contrast, in Balb/C mice perfusion recovery is limited and necrosis is common. Utilizing both experimental and computational approaches we identified mir- 93 as the miR that was MOST differentially expressed based on the combination of strain and ischemia. miR- 93 was significantly increased following HLI (p<0.0001 and >4-fold increase) in B6 mice not in Bab/C. Local delivery of a pre-miR93 led to greater perfusion recovery in Balb/C mice and systemic delivery of a miR-93 antagomer impaired perfusion recovery in C57/Bl6 mice. In-vitro, antagomers to miR-93 increased the extent of hypoxia induced apoptosis while treatment with pre-miR-93 reduced the extent of hypoxia-induced apoptosis in both cell types. Antagomers to miR-93 reduced, and administration of pre-miR 93 increased, proliferation. We consistently found that the cycle genes p53, E2F1, and p21 were altered. Collectively, central hypothesis that miR-93, can serve as a potent modulator of post-natal angiogenesis in PAD though its ability to regulate cycle genes alone, or with other potential targets. Specific Aim 1): Define the role that miR-93 plays in-vivo in regulating the extent of blood flow recovery post-HLI using gain of function and loss of function approaches by the examination of mice at appropriate pre-selected time points. Specific Aim 2): Utilize in-vitro studies to determine the extent to which th gene expression changes induced by miR-93 can be directly linked to miR-93/mRNA interactions and establish the extent to which the gene expression changes induced by miR-93 are cell and condition specific. Specific Aim 3): Measure miR-93 in an established bank of human plasma and skeletal human samples from patients with varying severity of PAD as well as a group of non-PAD controls.