miR-615, AKT/eNOS signaling, and angiogenesis

缺血性心血管疾病（CVD），由于动脉粥样硬化闭塞的动脉心脏，腿部，或 在美国，脑与相当大的发病率、死亡率和卫生保健支出有关。 新血管的诱导和编排对于响应损伤的组织修复至关重要， 心肌梗死或外周动脉疾病（PAD）。在对促血管生成刺激的反应中， 内皮细胞（EC）被激活以迁移和增殖形成初级毛细血管。但尽管 由于内皮细胞在新生血管生成中的重要性，我们对调节这一过程的机制的理解仍然存在 不太了解。新的研究表明，血管生成生长因子不能刺激 血管生成可能是由于受损的血管生成信号而不是由于这些生长因子的缺乏。 microRNAs（miRNAs）是一类小分子单链非编码RNA，能够抑制基因表达， 通过与mRNA靶的3'非翻译区（3'-UTR）的碱基配对来表达，并参与mRNA靶的3'非翻译区（3'-UTR）的表达。 心血管生物学中的各种病理生理过程，尽管它们在血管生成和 血管生成信号通路仍然不清楚。我们采用了微阵列分析方法， 从缺血性CVD受试者的血浆中，并确定miR-615- 5 p表达因缺血而增加 并在对促血管生成刺激的反应中减少-在小鼠和人中均重现了观察结果 体内缺血范例。初步和已发表的功能获得和丧失研究表明，miR-615- 5 p 过表达显著损害EC增殖、迁移和基质胶中网状管的形成，而 阻断miR-615- 5 p具有相反的效果。从机制上讲，使用无偏见的转录组学分析，我们 发现miR-615- 5 p抑制EC增殖，并与两个独特的靶点RASSF 2和IGF 2结合， 3 '-UTR并减少其表达，这种作用选择性地调节AKT/eNOS信号通路， EC。最后，全身静脉注射miR-615- 5 p抑制剂增加了血管形成 与接受了抗抑郁药的小鼠相比， 乱序对照抗miR注射。这些观察结果为中心假设提供了基础 miR-615- 5 p可能是EC增殖和血管生成反应的关键调节因子。更好地 为了了解miR-615- 5 p在AKT/eNOS信号传导和血管生成中的确切作用，我们将在Aim 1中描述miR-615- 5 p在AKT/eNOS信号传导和血管生成中的作用。 调控miR-615- 5 p在EC中表达的上游机制。在Aim 2中，我们将确定分子 这是miR-615- 5 p调节AKT/eNOS信号传导和EC功能的能力的基础，这些功能对血管生成至关重要。在 目的3，我们将探讨改变微血管中miR-615- 5 p表达对急性和慢性胰腺炎的影响。 慢性实验性缺血性损伤这些研究的结果将提供有关miR-615- 5 p的见解， 在EC生物学、病理生理学血管生成和心血管缺血状态中起作用，并可提供 新的目标，以挽救受损的血管生成信号的缺血性心血管疾病状态的范围。

Ischemic cardiovascular disease (CVD) due to atherosclerotic occlusion of the arteries to the heart, legs, or brain is associated with considerable morbidity, mortality, and health care expenditure in the United States. The induction and orchestration of new blood vessels is critical for tissue repair in response to injury such as myocardial infarction or peripheral artery disease (PAD). In response to pro-angiogenic stimuli, vascular endothelial cells (ECs) are activated to migrate and proliferate to form primary capillaries. However, despite the importance of ECs in neoangiogenesis, our understanding of the mechanisms regulating this process remains poorly understood. Emerging studies indicate that the inability of angiogenic growth factors to stimulate angiogenesis is likely due to impaired angiogenic signaling and not due to deficiency in these growth factors. MicroRNAs (miRNAs) are small, single-stranded, non-coding RNAs capable of repressing gene expression by base pairing to the 3' untranslated regions (3'-UTRs) of mRNA targets and are involved in a variety of pathophysiological processes in cardiovascular biology, though their function in angiogenesis and angiogenic signaling pathways remains poorly defined. We undertook a microarray profiling approach of plasma from subjects with ischemic CVD and identified that miR-615-5p expression is increased by ischemia and reduced in response to pro-angiogenic stimuli–observations that are recapitulated in both mice and human ischemic paradigms in vivo. Preliminary and published gain and loss-of-function studies reveal that miR-615-5p overexpression markedly impaired EC proliferation, migration, and network tube formation in matrigel, whereas blockade of miR-615-5p had the opposite effects. Mechanistically, using unbiased transcriptomic profiling, we find that miR-615-5p suppressed EC proliferation and binding to 2 unique targets–RASSF2 and IGF2–in their 3'-UTRs and reduced their expression, an effect that selectively regulated the AKT/eNOS signaling pathway in ECs. Finally, systemic intravenous administration of miR-615-5p inhibitors increased blood vessel formation and reduced infarct size and improved blood flow recovery in ischemic legs compared to mice that received scrambled control anti-miR injections. These observations provide the foundation for the central hypothesis that miR-615-5p may serve as a critical regulator of EC proliferation and angiogenic responses. To better understand the precise role of miR-615-5p in AKT/eNOS signaling and angiogenesis, we will in Aim1 delineate the upstream mechanisms governing miR-615-5p expression in ECs. In Aim2, we will determine the molecular basis for miR-615-5p's ability to regulate AKT/eNOS signaling and EC functions critical to angiogenesis. In Aim3, we will explore the effect of altering miR-615-5p expression in the microvasculature on acute and chronic experimental ischemic injury. The results of these studies will provide insights regarding miR-615-5p function in EC biology, pathophysiological angiogenesis, and cardiovascular ischemic states and may provide new targets to rescue impaired angiogenic signaling for a range of ischemic cardiovascular disease states.