PlGF-HIF1a-miRNA Axis in Sickle Pulmonary Hypertension

项目摘要 肺动脉高压（PHT）发生在约30%的镰状细胞贫血（SCA）患者中， 诊断后2年内死亡。这种血管病变的发病机制可能是多因素的， 溶血诱导的一氧化氮生物利用度受损，促凝血剂引起的慢性血栓栓塞 状态，并增加内皮素-1（ET-1）。我们的研究表明，胎盘生长因子（PlGF）是一种 镰状红细胞产生大量的血管生成生长因子，诱导表达强效的 肺血管收缩剂ET-1和促凝血剂纤溶酶原激活物抑制剂PAI-1 肺微血管内皮细胞（HPMVEC）。PlGF通过抑制ET-1和PAI-1的表达而增加ET-1和PAI-1的表达。 诱导缺氧诱导因子-1（HIF-1）。异位PlGF可在实验上诱发PHT 在以ET-1增加为特征的正常小鼠中的表达，如在转基因镰状小鼠中观察到的， SCA患者。我们最近观察到，在HPMVEC中，PlGF介导的HIF-1和PAI-1的诱导作用是在 在转录水平上，它们由三种特定的microRNA（miRs）调控。关于后记，人们知之甚少。 HIF-1a、ET-1和PAI-1或RNA结合蛋白的转录、miR介导的调节表达 稳定这些促进PHT基因的mRNA。因此，我们的总体假设是， RNA结合蛋白和miRNAs改变HIF-1、ET-1和PAI-1 mRNA的稳定性，并直接影响HIF-1、ET-1和PAI-1的表达。 参与PHT的发展。为了解决这个假设，在目标1中，我们将确定后- 调节PlGF介导的HIF-1a、ET-1和PAI-1表达的转录机制，并鉴定 RNA结合蛋白和参与结合mRNA 3'UTR从而调节mRNA翻译的特异性miR HIF-1a、ET-1和PAI-1 mRNA。在目标2中，我们将确定调节PAI-1的miR基因是否 SKA2基因是从NFYC共合成的， SKA2初级转录物，或从较小的前体miRNA转录单位独立转录。在aim中 3，我们将证明这些miRs在HIF-1、ET-1和PAI-1的遗传调节中的需要， 过表达PlGF并发展PHT的小鼠模型。最后，我们将确定等离子体的关联 在有和没有PHT症状的SCA患者中，这些miRNA水平与血浆PlGF、ET-1和PAI-1相关。 这些研究将推进我们对RNA结合蛋白和miR如何调节一些关键的细胞因子的认识。 参与镰状PHT的基因，以及这些miRNA的表达本身是如何调节的。这些研究可能会 提供了用于评估PHT的新的诊断生物标志物，以及用于 目前几乎没有或没有治疗选择。

Project Summary Pulmonary hypertension (PHT) occurs in ~30% of patients with sickle cell anemia (SCA) and results in ~50% mortality within 2 years of diagnosis. The pathogenesis of this vasculopathy is likely multi-factorial, potentiated by hemolysis-induced impaired nitric oxide bioavailability, chronic thrombo-embolism from a procoagulant state, and increased endothelin-1 (ET-1). Our studies have shown that placenta growth factor (PlGF), an angiogenic growth factor produced in high amounts by sickle erythroid cells, induces expression of the potent pulmonary vasoconstrictor ET-1, and a procoagulant, plasminogen activator inhibitor-1 (PAI-1), from human pulmonary microvascular endothelial cells (HPMVEC). PlGF increases ET-1 and PAI-1 expression via induction of hypoxia-inducible factor-1¿ (HIF-1¿). PHT can be induced experimentally by ectopic PlGF expression in normal mice characterized by increased ET-1, as is observed in transgenic sickle mice and in SCA patients. We recently observed that PlGF-mediated induction of HIF-1¿ and PAI-1 in HPMVEC is post- transcriptionally regulated by three specific microRNAs (miRs). Relatively little is known of the post- transcriptional, miR-mediated, regulated expression of HIF-1a, ET-1 and PAI-1, or of the RNA-binding proteins that stabilize the mRNAs of these genes that promote PHT. Thus our overall hypothesis is that cytoplasmic RNA-binding proteins and miRNAs alter the stability of HIF-1¿, ET-1, and PAI-1 mRNAs, and are directly involved in the development of PHT. To address this hypothesis, in Aim 1, we will determine the post- transcriptional mechanisms which regulate PlGF-mediated expression of HIF-1a, ET-1 and PAI-1 and identify RNA binding proteins and the specific miRs involved in binding to mRNA 3'UTRs thus regulating translation of HIF-1a, ET-1, and PAI-1 mRNAs. In Aim 2, we will determine whether the genes for miRs that regulate PAI-1 expression, and are located within introns of NFYC and SKA2 genes are co-synthesized from the NFYC and SKA2 primary transcripts, or are independently transcribed from a smaller, pre-miRNA transcription unit. In Aim 3, we will demonstrate the requirement of these miRs in the regulation of HIF-1¿, ET-1 and PAI-1 in genetic mouse models that over-express PlGF and develop PHT. Finally, we will determine the association of plasma levels of these miRNAs to plasma PlGF, ET-1 and PAI-1 in SCA patients with and without PHT symptoms. These studies will advance our knowledge as to how RNA binding proteins and miRs regulate some of the key genes involved in sickle PHT, and how expression of these miRNAs is itself regulated. These studies will likely provide new diagnostic bio-markers for assessment of PHT, and novel therapeutic targets for a disease that currently has few or no therapeutic options.