PlGF-HIF1a-miRNA Axis in Sickle Pulmonary Hypertension

项目摘要 约30%的镰状细胞性贫血(SCA)患者会出现肺动脉高压(PHT)，约50%的患者会导致肺动脉高压 确诊后2年内死亡。这种血管病变的发病机制可能是多因素共同作用的。 通过溶血导致一氧化氮生物利用度受损，促凝剂导致的慢性血栓栓塞症 内皮素-1(ET-1)水平升高。我们的研究表明，胎盘生长因子(PlGF)和 镰状红系细胞大量产生血管生成生长因子诱导表达 肺血管收缩因子ET-1和纤溶酶原激活物抑制物-1(PAI-1) 肺微血管内皮细胞(HPMVEC)。PlGF通过促进ET-1和PAI-1的表达 缺氧诱导因子-1(HIF-1)的诱导。异位PlGF可在实验上诱导PHT 以ET-1升高为特征的正常小鼠的表达，就像在转基因镰刀鼠和 SCA患者。我们最近观察到，PlGF介导的HPMVEC中HIF-1和PAI-1的诱导是后 受三个特定的microRNAs(MiRs)转录调控。人们对这一职位知之甚少- MiR介导的HIF-1a、ET-1和PAI-1或RNA结合蛋白的转录调控表达 这稳定了这些促进PHT的基因的mRNAs。因此，我们的总体假设是细胞质 RNA结合蛋白和miRNAs改变HIF-1、ET-1和PAI-1 mRNAs的稳定性，并直接 参与了PHT的开发。为了解决这一假设，在目标1中，我们将确定- 调节PlGF介导的HIF-1a、ET-1和PAI-1表达的转录机制及鉴定 RNA结合蛋白和参与与mRNA3‘UTRs结合从而调节翻译的特定miR HIF-1a、ET-1和PAI-1mRNAs。在目标2中，我们将确定调节PAI-1的miRs的基因 表达，并位于NFYC和SKA2基因的内含子内，由NFYC和SKA2共同合成 SKA2初级转录本，或从较小的前miRNA转录单位独立转录而来。在AIM 3，我们将论证这些MIR在HIF-1β、ET-1和PAI-1的基因调控中的需求 过度表达PlGF并发展为PHT的小鼠模型。最后，我们将确定血浆与 伴有和不伴有PHT症状的SCA患者血浆PlGF、ET-1和PAI-1水平的变化。 这些研究将促进我们对RNA结合蛋白和miRs如何调节一些关键蛋白的了解 参与镰状PHT的基因，以及这些miRNAs的表达本身是如何调控的。这些研究很可能 为评估PHT提供新的诊断生物标志物，并为一种疾病提供新的治疗靶点 目前几乎没有治疗选择。

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.