Role of heparin binding growth factors in vascular leakage and fatal bleeding

描述（由申请方提供）：关于循环肝素结合血管生成生长因子在危重儿童中肝素样药物或类肝素诱导的出血性疾病发病机制中的作用，目前知之甚少。我们已经发现成纤维细胞生长因子-2（FGF-2），一种血管生成肝素结合生长因子（HBGF），在肝素和戊聚糖多硫酸酯（一种肝素样药物）诱导的出血性疾病的发病机制中的新作用。其他人已经发现，在鸡CAM测定中，增强FGF-2活性的FGF结合蛋白（BP-1）诱导血管渗漏和出血。基于这些发现，我们假设FGF-2单独或与BP-1或VEGF-A组合，增强肝素样药物诱导的毛细血管通透性变化，并增加其引起致命性血管病变的风险。将在三个具体目标中检验这一假设：目的1将首先定义FGF-2、BP-1、VEGF-A和从重症儿童采集的血浆尿液样品单独或与肝素组合增加培养的人微血管内皮细胞（HMVEC）的渗透性的基本信号传导途径，并确定VEGFR-2、Tie-2、Src、和Rho激酶通路的作用。其次，从接受或不接受肝素治疗的FGF-2血浆水平较高的重症儿童中采集的血浆/尿液样本将用于探索类似的途径，并开发HMVEC渗透性生物测定法，以发现新的生物标志物，从而识别接受肝素治疗时存在出血风险的儿童。在目的2中，tet诱导型Tg小鼠将用于确定BP-1在这些出血性疾病的发病机制中的相对贡献。此外，来自TVA-Tg小鼠的内皮细胞将用血管生成逆转录病毒载体（RCAS）转导，以增强FGF-2和其他HBGF向循环中的释放，并确定它们在类肝素诱导的血管病变发病机制中的作用。目的3将验证血管生成素-1（Angiopoietin-1，Ang-1），一种血管生成抗渗透性生长因子，和FGF-2受体酪氨酸激酶抑制剂PD 173074，将改善类肝素和FGF-2诱导的小鼠血管畸形的临床结果的假设。Rho激酶途径在这一过程中的相对贡献和作用也将被探讨。这些实验将产生与释放到危重患儿循环中的HBGF在肝素诱导的血管栓塞的临床结局中所起作用相关的新知识，并测试抗渗透性治疗将改善这些儿童临床结局的新概念。公共卫生相关性：该提案将检验以下假设：释放到危重患儿循环中的肝素结合生长因子在肝素样药物诱导的血管渗漏和致死性血管病变的发展中起关键作用。此外，我们将在野生型和转基因小鼠中测试抗渗透性疗法将改善肝素样药物诱导的致死性脑血管病的临床结果的新概念。这些发现可能对需要使用肝素（心脏搭桥、ECMO和血液透析）的接受体外程序的危重儿童以及患有血管肿瘤和其他血管生成性疾病的幼儿的治疗具有更广泛的临床意义。

DESCRIPTION (provided by applicant): Very little is known about the role that circulating heparin binding angiogenic growth factors play in the pathogenesis of bleeding disorders induced by heparin-like drugs or heparinoids in critically ill children. We have found a new role for Fibroblast Growth Factor -2 (FGF-2), an angiogenic heparin binding growth factor (HBGF), in the pathogenesis of bleeding disorders induced by heparin and pentosan polysulfate, an heparin-like drug. Others have found that an FGF binding protein (BP-1) that enhances the activity of FGF-2 induces vascular leakage and bleeding in the chicken CAM assay. Based on these findings, we hypothesize that FGF-2, alone or in combination with BP-1 or VEGF-A, enhances the capillary permeability changes induced by heparin-like drugs and increases their risk of causing lethal hemorrhages. This hypothesis will be tested in three specific aims: Aim 1 will define first the basic signaling pathways by which FGF-2, BP-1, VEGF-A, and plasma urine samples harvested from critically ill children, alone or in combination with heparin, increase the permeability of cultured human microvascular endothelial cells (HMVEC), and determine the relative contribution of the VEGFR-2, Tie-2, Src, and Rho kinase pathways in this process. Second, plasma/urine samples harvested from critically ill children with high plasma levels of FGF-2 treated with or without heparin, will be used to explore similar pathways and develop an HMVEC-permeability bioassay to discover new biomarkers to identify children at risk of bleeding when treated with heparin. In aim 2 tet-inducible Tg mice will be used to determine the relative contribution of BP-1 in the pathogenesis of these bleeding disorders. In addition, endothelial cells from TVA-Tg mice will be transduced with angiogenic retroviral vectors (RCAS), to enhance the release of FGF-2 and other HBGF into the circulation, and define their role in the pathogenesis of hemorrhages induced by heparinoids. Aim 3 will test the hypothesis that Angiopoietin-1 (Ang-1), an angiogenic anti-permeability growth factor, and the FGF-2 receptor tyrosine kinase inhibitor PD173074, will improve the clinical outcome of hemorrhages induced by heparinoids and FGF-2 in mice. The relative contribution and role of the Rho kinase pathway in this process will be explored as well. These experiments will generate new knowledge related to the role that HBGF released into the circulation of critically ill children play in the clinical outcome of hemorrhages induced by heparin, and test the novel concept that anti- permeability therapies will improve the clinical outcome of these children. PUBLIC HEALTH RELEVANCE: This proposal will test the hypothesis that heparin binding growth factors released into the circulation of critically ill children play a critical role in the development of vascular leakage and lethal hemorrhages induced by heparin-like drugs. In addition, we will test the novel concept that anti-permeability therapies will improve the clinical outcome of lethal hemorrhages induced by heparin-like drugs in in wild type and transgenic mice. These findings could have wider clinical implications for the treatment of critically ill children subjected to extracorporeal procedures) that required the use of heparin (cardiac bypass, ECMO, and hemodialysis and young children with vascular tumors and other angiogenic diseases.