BMP Signaling and Pulmonary Vasoreactivity

描述（由申请人提供）：家族性肺动脉高压（FPAH）患者遗传BMP 2型受体BMPR2的杂合突变。目前尚不清楚这些突变如何引起肺动脉高压（PH）。这些突变中有一半逃避无义介导的mRNA衰变并表达BMPR2突变mRNA （NMD阴性）。其他的是NMD阳性，不表达突变产物。由于NMD阴性突变表达突变基因产物，它们可能对肺血管系统发挥显性抑制作用。最近的证据支持这一点：NMD阴性与NMD阳性BMPR2突变的FPAH患者的诊断年龄更年轻。这表明NMD阴性突变会导致更严重的疾病。在携带不同Bmpr2突变的小鼠中进行的初步研究表明了这种情况发生的机制。杂合子Bmpr2外显子2缺失突变(Bmpr2？Ex2/+)比携带杂合Bmpr2+/-突变的小鼠PH值更严重。这与Bmpr2中eNOS活性降低和内皮细胞功能障碍有关。Ex2/+肺血管。eNOS过表达可逆转Bmpr2？Ex2/+突变小鼠，表明eNOS缺陷在加重ph中起关键作用。此外，Bmpr2？Ex2突变产物，通过异常的eNOS细胞内隔离干扰eNOS功能。由于在FPAH患者中也发现了类似的NMD阴性外显子2缺失突变，这些发现表明eNOS缺陷可能是NMD阴性BMPR2突变加剧FPAH患者PH的共同机制。我们提出了三个目标来检验这一假设，并定义这种情况发生的机制。目的1将比较肺血管Bmpr2的血管反应性、PH反应和eNOS活性。Ex2/+和Bmpr2+/-小鼠。Aims 2将使用细胞培养技术评估BMP信号如何正常调节eNOS在肺内皮细胞中的表达和活性，而Aims 3将比较eNOS在Bmpr2+/-和Bmpr2？Ex2/+小鼠，并将确定Bmpr2？Ex2/+突变产物对肺内皮细胞eNOS活性具有显性抑制作用。这些研究将深入了解NMD阳性FPAH BMPR2突变（如BMPR2 +/-突变）与NMD阴性突变(其表达和行为可能与BMPR2 ？Ex2/+突变)改变FPAH患者的血管反应。通过定义Bmpr2？Ex2抑制eNOS，它们也将为通过纠正FPAH中NMD阴性BMPR2突变体加工缺陷来改善PH的治疗方法奠定基础。

DESCRIPTION (provided by applicant): Patients with Familial Pulmonary Hypertension (FPAH) inherit heterozygous mutations in the BMP type 2 receptor BMPR2. It is unknown how these mutations cause pulmonary hypertension (PH). Half of these mutations escape Nonsense Mediated mRNA Decay and express BMPR2 mutant mRNA (NMD negative). Others are NMD positive and do not express mutant products. As NMD negative mutations express mutant gene products they may exert dominant inhibitory effects on the pulmonary vasculature. Recent evidence supports this: the age at diagnosis is younger in FPAH patients with NMD negative versus NMD positive BMPR2 mutations. This suggests NMD negative mutations cause more severe disease. Preliminary studies in mice carrying different Bmpr2 mutations suggest a mechanism by which this occurs. Mice with the heterozygous Bmpr2 Exon 2 deletion mutation (Bmpr2?Ex2/+) have more severe PH than mice carrying heterozygous null Bmpr2+/- mutations. This is associated with reduced eNOS activity and endothelial cell dysfunction in Bmpr2?Ex2/+ pulmonary vasculature. Over-expression of eNOS reverses the PH phenotype in Bmpr2?Ex2/+ mutant mice, indicating that eNOS deficiency plays a critical role in exacerbating PH. Furthermore, there is abnormal intracellular processing of the Bmpr2?Ex2 mutant product, and this interferes with eNOS function by abnormal intracellular sequestration of eNOS. As similar NMD negative Exon 2 deletion mutants are found in FPAH patients, these findings suggest that eNOS deficiency may be a common mechanism by which NMD negative BMPR2 mutations exacerbate PH in patients with FPAH. Three aims are proposed to test this hypothesis and define the mechanism by which this occurs. Aim 1 will compare vasoreactivity, PH responses and eNOS activity in the pulmonary vasculature of Bmpr2?Ex2/+ and Bmpr2+/- mice. Aims 2 will use cell culture techniques to evaluate how BMP signaling normally regulates eNOS expression and activity in pulmonary endothelial cells, while Aim 3 will compare eNOS regulation in Bmpr2+/- and Bmpr2?Ex2/+ mice, and will determine the molecular mechanism by which the Bmpr2?Ex2/+ mutant product exerts dominant inhibitory effects on eNOS activity in pulmonary endothelial cells. These studies will provide insight into how NMD positive FPAH BMPR2 mutations (like the Bmpr2+/- mutation) vs. NMD negative mutations (which are expressed and may behave like the Bmpr2?Ex2/+ mutation) modify vascular responses in patients with FPAH. By defining the mechanism by which Bmpr2?Ex2 inhibits eNOS they will also establish the foundation for therapeutic approaches to ameliorate PH by correcting NMD negative BMPR2 mutant processing defects in FPAH. PUBLIC HEALTH RELEVANCE: Pulmonary Arterial Hypertension is an invariably fatal disease for which there is no curative treatment. A major challenge for the scientific community therefore is to develop therapeutic approaches to prevent the progression of pulmonary vascular disease that develops in these patients. Our studies will provide important insight into a common pathway that regulates this process and identify approaches to interfere with this pathway and prevent the development of irreversible pulmonary vascular disease in these patients.