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，其特征是肺血管阻力持续升高，导致右室后负荷增加和右室肥厚，最终导致右室衰竭和死亡。肺血管重塑是PAH的重要病理特征，其主要原因是肺血管细胞增殖和抗凋亡能力增强。血管细胞表型异常与多种生长因子有关。生长因子与其受体酪氨酸激酶(RTK)结合，启动下游信号通路，导致细胞增殖和存活。因此，有效的治疗策略应该针对这些异常的血管细胞，从而干扰和逆转重建的肺血管系统。目前的血管扩张治疗主要提供症状缓解，而令人信服的证据表明，它们对严重重塑的血管具有直接的抗增殖/抗重塑作用，这一点在很大程度上还缺乏。在之前的资助期间，我们已经证明，伊马替尼抑制血小板衍生生长因子受体(PDGFR)可以逆转血管重塑，改善实验性PH患者的存活率。基于我们的工作，伊马替尼成功地在PAH中进行了临床开发，导致了积极的3期试验。然而，PDGF和生长因子介导的RTK信号转导途径的作用仍有待阐明。因此，该项目旨在a)利用激光辅助显微切割、实时聚合酶链式反应、原位杂交、免疫荧光和免疫组织化学等技术，分析生长因子及其信号分子(Src、PI3K、mTOR、JAK、ERK、P38、JNK、AKT、PLCy和PKC)在实验和人体组织中的特异性表达，b)利用小分子抑制剂和siRNA技术检测这些分子在原代血管细胞增殖和凋亡中的功能作用，C)通过转基因动物研究这些分子在PH发病机制中的细胞类型特异性(内皮细胞与平滑肌)的作用；d)评估RTK通路抑制剂在临床相关的PH动物模型中的治疗效果。将使用非侵入性成像技术(超声心动图、CT-FMT)、侵入性技术(米勒肺动脉导管)和组织形态计量学来评估PH。伊马替尼治疗的效果将通过不同的治疗模式(脉冲治疗和连续治疗)进行检验，此外还将在肺动脉狭窄模型中进行检验。还将探索通过吸入局部给予RTK的各种抑制剂的疗效。最后，将使用临床样本分析PAH患者对TK抑制剂(伊马替尼)个体反应的潜在指标/标记物。这个项目的总体目标是进一步发展这一概念，即通过靶向RTK信号通路，可以逆转/正常化PH中重构的血管结构。

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.