Regulation of Expression of Secreted Flt-1

描述（由申请人提供）：血管内皮生长因子（VEGF）是胚胎发育和成人血管稳态中新生血管形成的主要介质。VEGF有助于缺血组织中的适应性血管生成以及增殖性血管病变中的过度血管形成。因此，VEGF及其信号传导途径可以提供促进血管功能不全中的血管生成或抑制糖尿病视网膜病变或肿瘤中的血管生成的手段。对VEGF的反应由两种内皮细胞表面受体Fit-1和KDR介导。Flt-lmRNA的RNA加工变体产生分泌形式的Flt-l（“sFIt-1“），其以高亲和力结合VEGF并可抑制对VEGF的生物学应答。先前的研究表明，sFlt-1：FIt-1的比率在生理条件下可以变化，表明存在特异性控制导致sFlt-1的mRNA加工事件的分子机制。我们推测sFIt-1和Fit-1的相对表达是血管对VEGF反应性的重要生物学决定因素。拟议研究的目标是确定新的实验系统和生化检测，以发现sFIt-1表达的调控机制。我们的具体目标是：1）确定生物学模型适用于研究调节sFIt-1表达，2）确定参与Fit-1前mRNA加工的核因子，3）确定蛋白激酶C激活改变sFIt-1表达的转录后机制。在目标1中，我们将定量sFIt-1：Fit-1在血管发育和成人血管生成的细胞或动物模型中的表达。在目标2下，我们将使用无细胞系统探测特定和未知加工因子与Fit-1前mRNA的相互作用。在目的3中，我们将评估mRNA稳定性改变和前体mRNA加工在佛波酯诱导sFIt-1表达中的作用。这些研究将增加我们对适应性和治疗性血管生成机制的理解，并可能为开发新的药理学干预措施来调节sFlt-1表达，从而调节VEGF反应性提供理论基础。

DESCRIPTION (provided by applicant): Vascular endothelial growth factor (VEGF) is a major mediator of neovascularization in embryonic development and adult vascular homeostasis. VEGF contributes to adaptive angiogenesis in ischemic tissue as well as excessive vessel formation in proliferative vascular pathologies. VEGF and its signaling pathways thus may provide a means of either promoting angiogenesis in vascular insufficiency or inhibiting angiogenesis in diabetic retinopathy or in tumors. Responses to VEGF are mediated by two endothelial cell-surface receptors, Fit-1 and KDR. An RNA processing variant of Fit-1 mRNA produces a secreted form of Fit-1 ("sFIt-1 ") that binds VEGF with high affinity and can inhibit biological responses to VEGF. Previous studies indicate that the ratio of sFIt-l:FIt-1 can vary under physiological conditions, suggesting that molecular mechanisms exist to specifically control the mRNA processing events leading to sFIt-l. We postulate that the relative expression of sFIt-1 and Fit-1 is a significant biological determinant of vascular responsiveness to VEGF. The goals of the proposed research are to identify new experimental systems and biochemical assays to discover the regulatory mechanisms governing sFIt-1 expression. Our specific aims are 1) to identify biological models appropriate for the study of regulated sFIt-1 expression, 2) to identify nuclear factors involved in Fit-1 pre-mRNA processing, and 3) to determine the post-transcriptional mechanism by which protein kinase C activation alters sFIt-1 expression. In Aim 1, we will quantify sFIt-1:Fit-1 expression in cell- or animal-based models of vascular development and adult angiogenesis. Under Aim 2, we will probe the interaction of specific and unknown processing factors with Fit-1 pre-mRNA using cell-free systems. In Aim 3, we will assess the role of altered mRNA stability and pre-mRNA 3rocessing in the induction of sFIt-1 expression by phorbol ester. These studies will increase our understanding of the mechanisms of both adaptive and therapeutic angiogenesis, and may provide rationale for developing novel pharmacological interventions to modulate sFlt-1 expression and, thereby, VEGF responsiveness.