MiR-26a, endothelial cells, and neovascularization

描述(由申请人提供)：在美国，由于心脏、腿部或大脑动脉的动脉粥样硬化性闭塞而导致的缺血性心血管疾病与相当大的发病率、死亡率和医疗保健支出有关。新血管的诱导和协调对于应对心肌梗死或外周动脉疾病(PAD)等损伤的组织修复至关重要。作为对促血管生成刺激的反应，血管内皮细胞(ECs)被激活以迁移和增殖，形成初级毛细血管。然而，尽管内皮细胞在新生血管形成中的重要性，我们对调控这一过程的机制仍然知之甚少。MicroRNAs(MiRNAs)是一种小的、单链的、非编码的RNA，能够通过碱基配对的方式抑制基因的表达，参与心血管生物学中的各种病理生理过程，但它们在血管内皮细胞生长和血管生成中的作用尚不清楚。我们在内皮细胞中进行了微阵列分析方法，并发现促血管生成刺激，如血管内皮生长因子，降低了miR-26a的表达，而反血管生成刺激，如TSP-1，增加了miR-26a的表达--这一观察结果在小鼠和人类体内的缺血模型中都得到了概括。功能获得和功能丧失的研究表明，miR-26a过表达显著诱导细胞周期停滞，抑制迁移，减少促血管生成因子VEGF的释放，并损害基质中网络管的形成，而阻断miR-26a则具有相反的作用。从机制上讲，我们发现miR-26a通过与Smad1的3‘端非编码区结合并减少其表达而抑制EC的生长，这种作用降低了Id1的表达，增加了细胞周期抑制基因p21和p27的表达。最后，全身静脉注射miR-26a抑制剂增加了血管形成并减少了梗塞面积，相比于接受杂乱对照的antagomiR注射的小鼠。这些观察结果为中心假设提供了基础，即miR-26a可能作为EC生长和血管生成反应的关键调节因子。为了更好地了解miR-26a在BMP/Smad1信号和血管生成中的确切作用，我们提出了三个目标。在Aim1中，我们将描述调控内皮细胞miR-26a表达的上游机制。在AIM2中，我们将确定miR-26a调节BMP/Smad1信号和EC对血管生成至关重要的功能的分子基础。在AIM3中，我们将探讨miR-26a表达改变在急性和慢性实验性缺血性损伤中的作用。这些研究结果将为深入了解miR-26a在EC生物学、病理和生理血管生成以及心血管缺血状态中的作用提供新的靶点，并可能为促进缺血性心血管疾病的血管生成提供新的靶点。

DESCRIPTION (provided by applicant): Ischemic cardiovascular disease 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 injur 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. 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 vascular EC growth and angiogenesis remains poorly defined. We undertook a microarray profiling approach in endothelial cells and identified that pro-angiogenic stimuli, such as VEGF, decreased miR-26a expression, whereas anti-angiogenic stimuli, such as TSP-1, increased miR-26a expression-observations that are recapitulated in both mice and human ischemic paradigms in vivo. Gain and loss-of-function studies reveal that miR-26a overexpression markedly induced cell cycle arrest, inhibited migration, reduced the release of the pro-angiogenic factor VEGF, and impaired network tube formation in matrigel, whereas blockade of miR-26a had the opposite effects. Mechanistically, we find that miR-26a suppressed EC growth by binding uniquely to the 3'UTR of Smad1 and reduced its expression, an effect that decreased Id1 expression and increased cell cycle arrest genes p21 and p27 in ECs. Finally, systemic intravenous administration of miR-26a inhibitors increased blood vessel formation and reduced infarct size compared to mice that received scrambled control antagomiR injections. These observations provide the foundation for the central hypothesis that miR-26a may serve as a critical regulator of EC growth and angiogenic responses. To better understand the precise role of miR-26a in BMP/Smad1 signaling and angiogenesis, three aims are proposed. In Aim1, we will delineate the upstream mechanisms governing miR-26a expression in ECs. In Aim2, we will determine the molecular basis for miR-26a's ability to regulate BMP/Smad1 signaling and EC functions critical to angiogenesis. In Aim3, we will explore the effect of altered miR-26a expression on acute and chronic experimental ischemic injury. The results of these studies will provide insights regarding miR-26a function in EC biology, pathological and physiological angiogenesis, and cardiovascular ischemic states and may provide new targets to promote angiogenesis for ischemic cardiovascular disease.