Notch Function in Vascular Biology and Angiogenesis

描述（由申请人提供）：Notch通路是一种保守的信号传导机制，其功能是调节细胞命运的决定。本提案的总体目标是确定Notch在血管发育、生理性血管生成和血管系统维持过程中决定细胞命运的作用。我们的一般假设是Notch对动-静脉、静脉-淋巴发育和生理性血管生成至关重要。我们的总体策略将结合体外血管生成/淋巴管生成试验和小鼠模型来确定改变内皮细胞Notch活性的后果。我们使用体外实验进行的初步研究表明，Notch促进了血液内皮细胞的发芽和存活，但阻碍了毛细血管样脐带的形成。在Aim I中，通过体外血管生成实验，我们评估了Notch下游的信号机制，重点关注PI-3K/Akt和VEGF-C/VEGFR-3通路。在Aim II中，我们评估了VEGFR-3基因作为Notch的直接转录靶点，并通过操纵培养的淋巴内皮细胞探索了Notch在淋巴管生成中的作用。在Aim III中，我们使用小鼠模型激活或灭活胚胎脉管系统中的Notch来检测Notch功能。我们已经开发了小鼠模型，有条件地激活血管中的Notch信号，以确定Notch在胚胎发生的各个阶段的作用。Notch在胚胎血管系统中的激活将用于评估Notch在动脉/静脉和静脉/淋巴规范、血管平滑肌细胞募集、血管重塑和心脏发育中的作用。Notch基因在成人血管中表达，但其产后功能尚不清楚。在Aim IV中，Notch将在成人血管中有条件地激活，以评估血管完整性的功能。体内试验将检查Notch是否有助于或干扰VEGF或fgf诱导的血管生成。最后，Notch在卵巢中的功能将被研究，卵巢在卵泡发生和黄体形成过程中具有强大的生理性血管生成。利用垂体去切小鼠模型，允许激素诱导卵巢血管生成，我们将探索Notch在卵巢血管系统中的功能。这些小鼠将被用来确定当Notch在卵巢血管中有条件激活或使用Notch拮抗剂干扰时对卵巢血管生成的影响。我们的长期目标是了解Notch在人类血管疾病和病理性血管生成中的功能。

DESCRIPTION (provided by applicant): The Notch pathway is a conserved signaling mechanism that functions to modulate cell-fate decisions. The overall objectives of this proposal are to define roles for Notch in cell-fate determination during vascular development, physiological angiogenesis and maintenance of vasculature. Our general hypothesis is that Notch is critical for arterial-venous, venous-lymphatic development and for physiological angiogenesis. Our general strategy will use a combination of in vitro angiogenesis/lymphangiogenesis assays and mouse modeling to define the consequences of altering Notch activity in endothelial cells. Our preliminary studies using in vitro assays suggest that Notch promotes sprouting and survival of blood endothelial cells but blocks capillary-like cord formation. In Aim I, using in vitro angiogenesis assays, we evaluate signaling mechanisms that function downstream of Notch, with a focus on the PI-3K/Akt and VEGF-C/VEGFR-3 pathways. In Aim II, we evaluate the VEGFR-3 gene as a direct transcriptional target of Notch and explore a role for Notch in lymphangiogenesis by manipulating cultured lymphatic endothelial cells. In Aim III, we use mouse models that activate or inactivate Notch in embryonic vasculature to examine Notch function. We have developed mouse models that conditionally activate Notch signaling in the vasculature to define Notch action at various stages of embryogenesis. Notch activation in embryonic vasculature will be used to evaluate roles for Notch in arterial/venous and venous/lymphatic specification, recruitment of vascular smooth muscle cells, vascular remodeling, and heart development. Notch genes are expressed in adult vasculature but their postnatal function is unknown. In Aim IV, Notch will be conditionally activated in adult vasculature to evaluate function in vascular integrity. In vivo assays will examine whether Notch contributes to, or interferes, with VEGF- or FGF-induced angiogenesis. Finally, Notch function will be studied in the ovary, which has robust physiological angiogenesis during folliculogenesis and corpus luteum formation. Using a hypophysectomized mouse model that allows hormonal induction of ovarian angiogenesis, we will probe Notch function in ovarian vasculature. These mice will be used to define the consequences to ovarian angiogensis when Notch is conditionally activated in ovarian vessels or interfered with using Notch antagonists. Our long-term objective is to understand Notch function in human vascular disorders and during pathological angiogenesis.