Endothelin and EGFR Activation in Pulmonary Hypertension

描述（由申请人提供）：肺动脉高压（PH）的发展涉及血管平滑肌细胞（VSMC）增殖的改变。然而，其机制仍未得到解决。先前的数据表明，至少在一定程度上，PH的重塑是由内皮素-1 （ET-1）水平的增加和ET受体表达的改变（如ETA受体增加和ETB受体减少）引起的。我们的实验室之前已经证明，上调ETA信号可以刺激活性氧（ROS）的产生和VSMC的增殖。先前的研究表明，ros介导的表皮生长因子受体（EGFR）氧化诱导其共价二聚化。这种氧化二聚体EGFR被认为是不可逆交联的，具有组成活性。相反，一氧化氮（NO）的生物利用度在PH下降低，可能会降低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的硝化作用会抑制其活性。我们假设EGFR硝化在VSMC中的调节机制是通过ETB介导的位于VSMC层的NOS1的激活。进一步，我们假设ETB信号的缺失通过ETA导致EGFR激活导致ROS信号增强。为了在Aim 1中验证这些假设，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激活来刺激VSMC的生长，以及抗氧化剂和EGFR拮抗剂是否会减少这种作用。接下来，我们将过度表达ETB受体，以确定这是否会增强EGFR硝化，阻止ROS介导的EGFR激活，并导致VSMC生长停滞。为了证实异常EGFR信号在Aim 2中PH发展中的重要性，我们将在大鼠体内使用单芥碱注射诱导的PH模型。我们将确定在PH下EGFR的共价二聚化是否会增加，而EGFR硝化是否会减少。接下来，我们将确定EGFR拮抗剂是否会阻止与PH发展相关的VSMC增殖和血管重塑。最后，我们将确定通过电穿孔将ETB受体基因传递到大鼠肺是否会增加EGFR蛋白硝化，减少VSMC增殖，并缓解与PH进展相关的变化。

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.