Role of tyrosine kinases in the pathogenesis of pulmonary arterial hypertension

肺动脉高压（PAH）是一种进行性疾病，其特征为肺血管阻力持续升高，导致右心室（RV）后负荷增加和RV肥大，最终导致RV衰竭和死亡。肺血管重构是肺动脉高压的重要病理特征，其机制是肺血管细胞增殖增加和抗凋亡。几种生长因子与异常的血管细胞表型有关。生长因子与其受体酪氨酸激酶（RTK）结合并启动导致细胞增殖和存活的下游信号传导途径。因此，有效的治疗策略应该靶向这些异常的血管细胞，从而干扰和逆转重塑的肺血管系统。目前的血管舒张治疗主要提供症状缓解，并且在很大程度上缺乏其对严重重塑血管的直接抗增殖/抗重塑作用的令人信服的证据。在之前的资助期间，我们已经证明伊马替尼对血小板衍生生长因子受体（PDGFR）的抑制可逆转实验性PH中的血管重塑并改善生存率。基于我们的工作，伊马替尼成功地进行了PAH的临床开发，导致了积极的3期试验。然而，PDGF和生长因子介导的RTK信号传导的作用仍有待阐明。因此，该项目旨在a）分析生长因子及其信号分子的细胞特异性和隔室特异性表达（Src、PI3K、mTOR、JAK、ERK、P38、JNK、AKT、PLC γ和PKC），B）使用小分子抑制剂和siRNA技术检查这些分子对原代血管细胞增殖和凋亡的功能作用，C）研究细胞类型特异性（内皮与平滑肌）这些分子在PH发病机制中的作用，通过使用转基因动物和d）在PH的临床相关动物模型中评价RTK通路抑制剂的治疗功效。将使用非侵入性成像技术（超声心动图、CT-FMT）、侵入性技术（Millar肺动脉导管）和组织形态计量学来评估PH。e）通过各种治疗模式（脉冲vs.连续）以及另外在肺动脉狭窄模型中检查伊马替尼治疗的效果。还将探索通过吸入局部施用的各种RTK抑制剂的功效。最后，f）将使用临床样本分析PAH患者对TK抑制剂（伊马替尼）个体反应性的潜在指标/标志物。该项目的总体目标是进一步发展PH中重塑的血管系统可以通过靶向RTK信号通路逆转/正常化的概念。

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by sustained elevation of pulmonary vascular resistance leading to increased right ventricular (RV) afterload and RV hypertrophy that culminate in RV failure and death. Pulmonary vascular remodeling, the key pathological feature of PAH, is attributable to the increased proliferation and resistance to apoptosis of pulmonary vascular cells. Several growth factors are implicated in the abnormal vascular cell phenotypes. Growth factors bind to their receptor tyrosine kinase (RTK) and initiate downstream signaling pathways that result in cell proliferation and survival. An effective therapeutic strategy should therefore target these abnormal vascular cells thereby interfering and reversing the remodeled pulmonary vasculature. Current vasodilatory therapies mainly provide symptomatic relief and convincing evidence of their direct anti-proliferative/anti-remodeling effects on severely remodeled vessels is largely missing. In the previous funding period, we have shown that inhibition of the platelet derived growth factor receptor (PDGFR) by imatinib reverses vascular remodeling and improved survival in experimental PH. Based on our work, imatinib successfully underwent clinical development in PAH which resulted in a positive phase 3 trial. However, the role of PDGF and growth factor-mediated RTK signaling has still to be elucidated. The project therefore aims to a) analyze the cell- and compartment-specific expression of growth factors and their signaling molecules (Src, PI3K, mTOR, JAK, ERK, P38, JNK, AKT, PLCy and PKC) in experimental and human tissues using techniques such as laser assisted microdissection, real-time PCR, in situ hybridization, immunofluorescence and immunohistochemistry, b) examine the functional role of these molecules on proliferation and apoptosis of primary vascular cells using small molecule inhibitors and siRNA techniques, c) investigate the cell type-specific (endothelial versus smooth-muscle) role of these molecules in PH pathogenesis by employing transgenic animals and d) evaluate the therapeutic efficacy of inhibitors of RTK pathways in clinically relevant animal models of PH. The PH will be assessed using non-invasive imaging techniques (echocardiography, CT-FMT), invasive techniques (Millar pulmonary artery catheter) and histomorphometry. The effects of imatinib therapy will be examined e) by various treatment modes (pulsed vs. continuous), and additionally in the model of pulmonary stenosis. The efficacy of various inhibitors of RTKs administered locally via inhalation will also be explored. Finally, f) the potential indicators/markers of individual responsiveness of PAH patients to TK inhibitors (imatinib) will be analyzed using clinical samples. The overall goal of this project is to further develop the concept that the remodeled vasculature in PH can be reversed/ normalized by targeting RTK signaling pathways.