Pulmonary Vascular-Right Ventricular Axis Research Program

描述（由申请人提供）：肺动脉高压（PAH）是一种以肺血流动力学和血管生长调节受损为特征的致命疾病；右心室衰竭是导致死亡的主要原因。我们的研究发现肺动脉高压患者肺血管内皮一氧化氮合酶（eNOS）活性丧失，一氧化氮缺乏导致缺氧诱导因子-1 （HIF-1）激活，导致糖酵解、增殖、抗凋亡的内皮细胞促进血管重构。eNOS活性丧失的机制尚不清楚。eNOS也存在于心肌细胞中，在心肌细胞中，eNOS合成NO具有心脏保护作用；但eNOS还没有在人类多环芳烃心脏中进行过研究。eNOS的激活需要苏氨酸495位点（T495）和丝氨酸1177位点（S1177）的酶去磷酸化，后者是由环磷酸腺苷(cAMP)/蛋白激酶途径介导的？肾上腺素能受体(？[图1]。因为?在所有心力衰竭中均发现AR下调和脱敏。AR在eNOS活性丧失及其对肺血管-右心室（RV）轴的影响。移植的人类多环芳烃心脏具有脱敏性和低？AR，并且我们首次表明，来自人类多环芳烃肺的肺动脉内皮细胞（PAEC）具有类似的低数量和对？初步数据显示？AR脱敏是由于磷酸酶对受体的去磷酸化不足，磷酸酶被磷脂酰肌醇3-激酶（PI3Kg）的过度激活所抑制。？由于磷酸化受体的细胞内循环有缺陷，AR数较低。与PI3K对磷酸酶的抑制一致，PAH PAEC和心肌细胞具有高水平的非活性pT495 eNOS，确定了在任何类型的激动剂刺激下NO生成减少的原因，即eNOS处于非活性状态。与内皮细胞一样，PAH患者的心脏NO缺乏与hif激活和糖酵解代谢增加有关，这可以通过糖酵解酶表达和葡萄糖类似物的摄取[18F]，通过正电子发射断层扫描（空腹FDG-PET）检测PAH患者。令人兴奋的初步研究表明，b受体阻滞剂可以逆转？AR功能障碍，恢复磷酸酶介导的eNOS激活，从而分别增加cAMP和NO的产生。因此，我们假设PAH患者的心肌细胞和内皮细胞存在相互关联的？AR功能和eNOS激活导致hif介导的血管和心肌细胞表型失调，所有这些都可以通过受体阻滞剂逆转。我们检验了这一假设，即PAEC来源于患者的肺，外植的人类心脏，并在一项机械的纵向研究中？PAH患者的AR阻断。

DESCRIPTION (provided by applicant): Pulmonary arterial hypertension (PAH) is a fatal disease characterized by impaired regulation of pulmonary hemodynamics and vascular growth; right ventricular failure is the leading cause of death. Our studies have identified loss of pulmonary vascular endothelial nitric oxide synthase (eNOS) activity in PAH, and that the NO deficiency leads to hypoxia-inducible factor-1 (HIF-1) activation, which results in glycolytic, proliferative, apoptosis-resistant endothelial cells that promote vascular remodeling. The mechanism(s) of eNOS activity loss is unknown. eNOS is also present in the cardiomyocyte, where NO synthesis by eNOS is cardioprotective; but eNOS has not been studied in the human PAH heart. Activation of eNOS requires enzyme dephosphorylation at Threonine 495 (T495) and phosphorylation at Serine 1177 (S1177), the latter mediated by cyclic adenosine monophosphate (cAMP)/protein kinase pathways downstream of ?-Adrenergic Receptors (?AR) [Fig 1]. Because ?AR downregulation and desensitization are found in all heart failure, we investigated ?AR in the eNOS activity loss and its consequences on the pulmonary vascular-right ventricular (RV) axis. Explanted human PAH hearts have desensitization and low numbers of ?AR, and for the first time, we show that pulmonary arterial endothelial cells (PAEC) from human PAH lungs have similarly low numbers and de- sensitization of ?AR. Preliminary data reveal that ?AR desensitization is due to deficient dephosphorylation of the receptor by phosphatases, which are inhibited by over-activation of phosphatidyl inositol 3-kinase (PI3Kg). ?AR numbers are low due to defective intracellular recycling of phosphorylated receptors. Consistent with PI3K inhibition of phosphatases, PAH PAEC and cardiomyocytes have high levels of the inactive pT495 eNOS, identifying the cause of decreased NO production upon any type of agonist stimulation, i.e. eNOS is in an inactive state. As in the endothelial cell, NO deficiency in PAH hearts is associated with greater HIF-activation and increased glycolytic metabolism as identified by glycolytic enzyme expression and uptake of the glucose analogue [18F]2-fluoro-2-deoxy-D-glucose by positron emission tomography (fasting FDG-PET) in PAH patients. Exciting preliminary studies indicate that b-blockers reverse ?AR dysfunction, restore phosphatase- mediated eNOS activation, and thus increase cAMP and NO production, respectively. Thus, we hypothesize that cardiomyocytes and endothelial cells in PAH suffer from interconnected abnormalities of ?AR function and eNOS activation that lead to a HIF-mediated maladaptive vascular and cardiomyocyte phenotype, all of which is reversible by ?-blocker. We test this hypothesis PAEC derived from patients' lungs, explanted human hearts, and in a mechanistic longitudinal study of ?AR blockade in PAH patients.