Pulmonary Vascular-Right Ventricular Axis Research Program

描述(申请人提供)：肺动脉高压(PAH)是一种致命的疾病，其特征是肺血流动力学和血管生长调节受损；右心衰竭是主要的死亡原因。我们的研究证实，PAH时肺血管内皮型一氧化氮合酶(ENOS)活性丧失，NO缺乏导致缺氧诱导因子-1(HIF-1)激活，从而导致糖酵解、增殖、抗凋亡的内皮细胞促进血管重塑。ENOS活性丧失的机制(S)尚不清楚。ENOS也存在于心肌细胞中，在心肌细胞中，eNOS的合成不具有心脏保护作用；但eNOS在人类PAH心脏中尚未被研究。ENOS的激活需要苏氨酸495(T495)和丝氨酸1177(S1177)的酶去磷酸化，后者由β-肾上腺素能受体(？AR)下游的环磷酸腺苷(CAMP)/蛋白激酶通路介导[图1]。由于？AR下调和脱敏在所有心力衰竭中都被发现，我们研究了？AR在eNOS活性丧失中的作用及其对肺血管-右室(RV)轴的影响。移植的人PAH心脏具有脱敏和低数量的？AR，我们首次发现来自人PAH肺的肺动脉内皮细胞(PAEC)具有同样的低数量和对？AR的脱敏。初步数据表明，AR脱敏是由于磷脂酰肌醇3-激酶(PI3Kg)过度激活抑制的磷酸酶对受体的去磷酸化作用。？AR数低是由于磷酸化受体在细胞内的循环有缺陷。与PI3K抑制磷酸酶一致，PAH PAEC和心肌细胞有高水平的非活性pT495 eNOS，确定了在任何类型的激动剂刺激下NO产生减少的原因，即eNOS处于非活动状态。与内皮细胞一样，PAH患者心脏中的NO缺乏与更大的HIF激活和糖酵解代谢增加有关，这是通过PAH患者的糖酵解酶表达和摄取葡萄糖类似物[18F]2-氟-2-脱氧-D-葡萄糖(空腹FDG-PET)来确定的。令人兴奋的初步研究表明，b-受体阻滞剂逆转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中的心肌细胞和血管内皮细胞存在？AR功能和eNOS激活的相互关联的异常，导致HIF介导的血管和心肌细胞表型异常，所有这些都是被？-受体阻滞剂逆转的。我们验证了这一假设，PAEC来源于患者的肺、移植的人的心脏，并在一项对PAH患者的？AR阻断的机械性纵向研究中。

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.