PlGF-HIF1a-miRNA Axis in Sickle Pulmonary Hypertension

描述（由申请人提供）：肺高压（PHT）发生在约30%的镰状细胞性贫血（SCA）患者中，并导致约50%的患者在诊断后2年内死亡。这种血管病变的发病机制可能是多因素的，由溶血引起的一氧化氮生物利用度受损、促凝状态引起的慢性血栓栓塞和内皮素-1 （ET-1）升高等因素增强。我们的研究表明，胎盘生长因子（PlGF）是一种由镰状红细胞大量产生的血管生成生长因子，可诱导人肺微血管内皮细胞（HPMVEC）表达强效肺血管收缩因子ET-1和促凝剂纤溶酶原激活物抑制剂-1 （PAI-1）。PlGF通过诱导缺氧诱导因子-11 （HIF-11）增加ET-1和PAI-1的表达。在以ET-1升高为特征的正常小鼠中，实验中可以通过异位PlGF表达诱导PHT，在转基因镰状小鼠和SCA患者中也观察到这一点。我们最近观察到plgf介导的HIF-11和PAI-1在HPMVEC中的诱导受三种特异性microrna （miRs）的转录后调控。对于mir介导的转录后HIF-1a、ET-1和PAI-1的调控表达，以及稳定这些促进PHT的基因mrna的rna结合蛋白，我们所知相对较少。因此，我们的总体假设是细胞质rna结合蛋白和mirna改变了HIF-11、ET-1和PAI-1 mrna的稳定性，并直接参与了PHT的发展。为了解决这一假设，在Aim 1中，我们将确定调节plgf介导的HIF-1a、ET-1和PAI-1表达的转录后机制，并鉴定RNA结合蛋白和参与与mRNA 3' utr结合的特异性miRs，从而调节HIF-1a、ET-1和PAI-1 mRNA的翻译。在Aim 2中，我们将确定位于NFYC和SKA2基因内含子内的调控PAI-1表达的miRs基因是由NFYC和SKA2初级转录物共同合成的，还是由较小的前mirna转录单元独立转录的。在Aim 3中，我们将证明这些miRs在过度表达PlGF并发生PHT的遗传小鼠模型中调控HIF-11、ET-1和PAI-1的必要性。最后，我们将确定有或无PHT症状的SCA患者血浆中这些mirna水平与血浆PlGF、ET-1和PAI-1的关系。这些研究将促进我们了解RNA结合蛋白和mirna如何调节镰状PHT中涉及的一些关键基因，以及这些mirna的表达如何被自身调节。这些研究可能会为PHT的评估提供新的诊断生物标志物，并为目前很少或没有治疗选择的疾病提供新的治疗靶点。

DESCRIPTION (provided by applicant): 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-11 (HIF-11). 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-11 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-11, 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-11, 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. PUBLIC HEALTH RELEVANCE: Pulmonary hypertension (PHT) is associated with high death rate among sickle cell anemia patients. We have found that genes (e.g. endothelin-1) associated with PHT are increased in these patients, and we propose to study specific microRNAs involved in their regulation.