MiR-26a, endothelial cells, and neovascularization

描述（由申请人提供）：在美国，由于通往心脏、腿部或大脑的动脉粥样硬化性闭塞导致的缺血性心血管疾病与相当高的发病率、死亡率和医疗保健支出相关。新血管的诱导和协调对于心肌梗死或外周动脉疾病（PAD）等损伤后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生成中的重要性，我们对这一过程的调节机制的理解仍然知之甚少。MicroRNAs （miRNAs）是一种小的单链非编码rna，能够通过与mRNA靶标的3‘非翻译区（3’-UTRs）的碱基配对来抑制基因表达，并参与心血管生物学中的多种病理生理过程，尽管它们在血管EC生长和血管生成中的功能尚不明确。我们在内皮细胞中采用了微阵列分析方法，发现促血管生成刺激，如VEGF，降低了miR-26a的表达，而抗血管生成刺激，如TSP-1，增加了miR-26a的表达，这些观察结果在小鼠和人类体内缺血范式中都得到了概括。功能增益和功能丧失研究表明，miR-26a过表达显著诱导细胞周期阻滞，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。在机制上，我们发现miR-26a通过唯一结合Smad1的3'UTR抑制EC生长并降低其表达，这种作用降低了EC中Id1的表达并增加了细胞周期阻滞基因p21和p27。最后，全身静脉注射miR-26a抑制剂增加了血管形成，减少了梗死面积，与接受混乱对照安塔哥米注射的小鼠相比。这些观察结果为中心假设提供了基础，即miR-26a可能是EC生长和血管生成反应的关键调节剂。为了更好地了解miR-26a在BMP/Smad1信号传导和血管生成中的确切作用，提出了三个目标。在Aim1中，我们将描述在内皮细胞中调控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功能的分子基础，这些功能对血管生成至关重要。在Aim3中，我们将探讨miR-26a表达改变对急慢性实验性缺血损伤的影响。这些研究结果将为miR-26a在EC生物学、病理生理性血管生成和心血管缺血状态中的功能提供新的见解，并可能为缺血性心血管疾病促进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.