Cellular Defects in Familial Pulmonary Hypertension

描述(申请人提供)：原发性肺动脉高压(PPH)是一种病因不明的罕见疾病，其特征是严重的孤立性肺动脉高压导致右心衰竭和死亡。家族性PPH(FPPH)患者的遗传学研究表明，大多数患者携带BMPII型受体(BMPR2)基因突变，而功能分析表明，散发性PPH患者也存在BMP信号转导缺陷。因此，对携带明确BMPR2突变的FPPH患者的细胞功能障碍的分析为探索散发性和家族性PPH潜在的病理事件提供了机会。尽管如此，这些突变导致的细胞缺陷基本上是未知的，因为直接评估细胞功能变化的方法受到限制，无法获得FPPH患者来源的血管细胞。为了解决这一问题，我们开发了一种替代系统，使用来自FPPH患者的原代皮肤成纤维细胞来研究不同的BMPR2突变对细胞功能的影响。这些细胞易于获取和繁殖，并且与血管平滑肌细胞具有共同的特性。来自FPPH患者的皮肤成纤维细胞和肺动脉平滑肌细胞对BMPs没有反应而增殖，并显示转化生长因子-β激活的Smads的结构性磷酸化。这些发现是与不同的BMPR2突变相关的一致异常的第一个证据。在这些研究中，我们将通过使用FPPH成纤维细胞和肺动脉平滑肌细胞来询问Smad信号中这种缺陷对功能的影响，从而验证这个细胞培养系统。然后，我们将使用这些方法来评估一种无偏见的方法，以确定携带一系列不同BMPR2突变的FPPH细胞共享的细胞蛋白质组的变化。这些研究将建立一个技术平台，以识别和评估这些患者和其他形式的肺动脉高压患者的细胞异常。

DESCRIPTION (provided by applicant): Primary pulmonary hypertension (PPH) is a rare disease of unknown etiology characterized by severe, isolated pulmonary arterial hypertension leading to right ventricular failure and death. Genetic studies in patients with Familial PPH (FPPH) indicate that the majority carry mutations in the BMP Type II receptor (BMPR2) locus, while functional assays indicate that defects in BMP-signaling are also found in patients with sporadic PPH. For this reason, analysis of cellular dysfunction in FPPH patients carrying defined BMPR2 mutations provides an opportunity to explore pathological events underlying both sporadic and familial forms of PPH. Despite this, the cellular defects resulting from these mutations are essentially unknown, as direct approaches to evaluate alterations in cell function have been hindered by limited access to FPPH patient derived vascular cells. To address this we have developed a surrogate system using primary skin fibroblasts from patients with FPPH to study effects of diverse BMPR2 mutations on cellular function. These cells are easy to harvest and propagate, and share properties in common with vascular smooth muscle cells. Skin fibroblasts and pulmonary artery smooth muscle cells from patients with FPPH do not proliferate in response to BMPs, and show constitutive phosphorylation of the TGF-beta-activated Smads. These findings are the first evidence of a consistent abnormality associated with diverse BMPR2 mutations. In these studies we will validate this cell culture system by interrogating the functional impact of this defect in Smad-signaling using FPPH fibroblasts and pulmonary artery smooth muscle cells. We will then use these to evaluate an unbiased approach to identify changes in the cellular proteome shared by FPPH cells carrying a range of different BMPR2 mutations. These studies will establish a technological platform to identify and evaluate cellular abnormalities in these patients and in patients with other forms of pulmonary arterial hypertension.