Endothelin and EGFR Activation in Pulmonary Hypertension

描述（由申请方提供）：肺动脉高压（PH）的发生涉及血管平滑肌细胞（VSMC）增殖的改变。然而，机制仍然没有解决。先前的数据已经表明PH中的重塑至少部分地由内皮素-1（ET-1）水平的增加和ET受体表达的改变引起，使得ETA受体增加而ETB受体减少。我们的实验室先前已经表明，上调的ETA信号刺激活性氧（ROS）的产生和VSMC增殖。以往的研究表明，ROS介导的表皮生长因子受体（EGFR）的氧化诱导其共价二聚化。这种氧化的二聚体EGFR被认为是不可逆交联的和组成型活性的。相反，一氧化氮（NO）的生物利用度降低，在PH潜在地减少过氧亚硝酸盐介导的酪氨酸硝化。这可能是重要的，因为已知EGFR的硝化抑制其活性。我们推测VSMC中EGFR硝化的调节机制是通过ETB介导的位于VSMC层的NOS 1的激活。此外，我们假设ETB信号传导的丧失通过ETA导致增强的ROS信号传导，从而导致EGFR活化。为了检验目标1中的这些假设，我们将使用VSMC来确定ET-1是否通过ROS介导的共价二聚化和EGFR活化来刺激VSMC生长，以及抗氧化剂和EGFR拮抗剂是否降低这种作用。接下来，我们将过表达ETB受体以确定这是否会增强EGFR硝化，防止ROS介导的EGFR活化，并导致VSMC生长停滞。为了证实异常EGFR信号传导在目的2中PH发展中的重要性，我们将利用在大鼠中注射野百合碱诱导的PH体内模型。我们将确定EGFR的共价二聚化是否在PH中增加，EGFR硝化是否减少。接下来，我们将确定是否EGFR拮抗剂将防止VSMC增殖和血管重塑与PH的发展。最后，我们将确定是否使用基因传递的ETB受体的大鼠肺使用电穿孔将增加EGFR蛋白硝化，减少VSMC增殖，并减轻与PH的进展相关的变化。 肺动脉高压（PH）是一种罕见但严重的疾病，主要影响年轻女性，并最终导致死亡。PH的中心病理事件是肺血管平滑肌细胞（VSMC）的不受控制的生长，导致疾病进一步进展。本申请旨在描述翻译后表皮生长因子受体（EGFR）修饰在VSMC生长和PH发展中的作用，并提出基于选择性VSMC内基因电穿孔的新方法的EGFR抑制疗法。

DESCRIPTION (provided by applicant): The development of pulmonary hypertension (PH) involves altered vascular smooth muscle cell (VSMC) proliferation. However, the mechanisms are still unresolved. Previous data have shown the remodeling in PH is induced, at least in part, by increased levels of endothelin-1 (ET-1) and alterations in the expression of ET receptors such that ETA receptors are increased and ETB receptors are diminished. Our lab has previously shown that up-regulated ETA signaling stimulates reactive oxygen species (ROS) generation and VSMC proliferation. Previous studies have shown that ROS-mediated oxidation of the epidermal growth factor receptor (EGFR) induces its covalent dimerization. This oxidized dimeric EGFR is thought to be irreversibly cross-linked and constitutively active. Conversely, nitric oxide (NO) bioavailability is diminished in PH potentially decreasing peroxynitrite mediated tyrosine nitration. This may be important as the nitration of EGFR known to inhibit its activity. We hypothesize that the mechanism by which EGFR nitration is regulated in VSMC is via ETB mediated activation of NOS1 located in the VSMC layer. Further we hypothesize that the loss of ETB signaling leads to enhanced ROS signaling through ETA leading to EGFR activation. To test these hypotheses in Aim 1 we will use VSMC to determine if ET-1 stimulates VSMC growth through ROS-mediated covalent dimerization and activation of EGFR and if antioxidants and EGFR antagonists reduce this effect. Next, we will over-express ETB receptors to determine if this will enhance EGFR nitration, prevent ROS- mediated activation of EGFR, and lead to VSMC growth arrest. To confirm the importance of aberrant EGFR signaling in the development of PH in Aim 2 we will utilize an in vivo model of PH induced by monocrotaline injection in the rat. We will determine if the covalent dimerization of EGFR is increased in PH and EGFR nitration is decreased. Next we will determine if EGFR antagonists will prevent the VSMC proliferation and vessel remodeling associated with the development of PH. Finally, we will determine if using gene delivery of the ETB receptor to the rat lung using electroporation will increase EGFR protein nitration, reduce VSMC proliferation, and alleviate the changes associated with progression of PH. PUBLIC HEALTH RELEVANCE: Pulmonary hypertension (PH) is a rare but severe disease mostly affecting young women and ultimately leading to death. The central pathological event of PH is uncontrolled growth of pulmonary vascular smooth muscle cells (VSMC) resulting in further disease progression. This application aimed to delineate the role of posttranslational epidermal growth factor receptor (EGFR) modifications in VSMC growth and PH development and suggests the therapy for EGFR inhibition based on the novel method of selective intra-VSMC gene electroporation.