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

描述(申请人提供)：目前对循环肝素结合血管生长因子在肝素类药物或肝素类药物引起的危重儿童出血性疾病的发病机制中所起的作用知之甚少。我们发现成纤维细胞生长因子-2(成纤维细胞生长因子-2)是一种血管生成肝素结合生长因子(HBGF)，在肝素和肝素类药物多聚硫酸戊聚糖引起的出血疾病的发病机制中发挥了新的作用。其他人发现，在鸡的CAM检测中，一种能增强成纤维细胞生长因子-2活性的成纤维细胞生长因子结合蛋白(BP-1)会导致血管渗漏和出血。基于这些发现，我们假设，成纤维细胞生长因子-2单独或与BP-1或血管内皮生长因子-A联合使用可增强肝素类药物引起的毛细血管通透性改变，并增加其导致致命性出血的风险。这一假设将在三个具体的目标中得到验证：目标1将首先确定基本的信号通路，通过这些信号通路，从危重患儿采集的血浆尿样以及单独或与肝素联合使用的尿样可增加培养的人微血管内皮细胞(HMVEC)的通透性，并确定VEGFR-2、Tie-2、Src和Rho激酶通路在这一过程中的相对贡献。其次，从接受或不接受肝素治疗的高血浆成纤维细胞生长因子-2水平的危重儿童身上采集的血浆/尿液样本，将用于探索类似的途径，并开发一种HMVEC通透性生物测定，以发现新的生物标志物，以确定使用肝素治疗时有出血风险的儿童。在AIM 2中，将使用TET诱导的TG小鼠来确定BP-1在这些出血性疾病的发病机制中的相对贡献。此外，TVA-TG小鼠的内皮细胞将被血管生成逆转录病毒载体(RCAS)转导，以促进成纤维细胞生长因子-2和其他HBGF进入循环，并确定它们在肝素诱导的出血发病机制中的作用。目的3验证血管生成抗通透性生长因子血管生成素-1(Ang-1)和成纤维细胞生长因子-2受体酪氨酸激酶抑制剂PD173074能改善肝素和成纤维细胞生长因子-2诱导的小鼠出血的临床转归的假说。此外，还将探讨Rho激酶通路在这一过程中的相对贡献和作用。这些实验将产生与HBGF释放到危重儿童血液循环中在肝素引起的出血的临床结果中所扮演的角色相关的新知识，并测试抗渗透疗法将改善这些儿童的临床结果的新概念。公共卫生相关性：这项提案将检验这样一种假设，即释放到危重儿童循环中的肝素结合生长因子在肝素类药物导致的血管渗漏和致命出血的发生中发挥关键作用。此外，我们还将在野生型和转基因小鼠身上测试抗渗透疗法将改善肝素类药物引起的致命性出血的临床结局这一新概念。这些发现可能对需要使用肝素(体外循环、体外循环、血液透析和血液透析)的危重儿童以及患有血管肿瘤和其他血管新生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.