Notch Function in Vascular Biology and Angiogenesis

描述(申请人提供)：Notch通路是一种保守的信号机制，其功能是调节细胞命运的决定。这项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对于动-静脉、静脉-淋巴的发育和生理性的血管生成是至关重要的。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。我们在体外的初步研究表明，Notch促进血管内皮细胞的萌发和存活，但阻止毛细血管样的脐带形成。在目的I中，利用体外血管生成试验，我们评估了Notch下游的信号机制，重点是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管内皮细胞来探索Notch在淋巴管生成中的作用。在AIM III中，我们使用胚胎血管系统中激活或失活Notch的小鼠模型来检测Notch的功能。我们已经开发了有条件地激活血管系统中的Notch信号的小鼠模型，以确定Notch在胚胎发育的不同阶段的作用。Notch在胚胎血管系统中的激活将被用于评估Notch在动脉/静脉和静脉/淋巴规范、血管平滑肌细胞的招募、血管重塑和心脏发育中的作用。Noch基因在成体血管中表达，但其出生后的功能尚不清楚。在AIM IV中，Notch将在成人血管系统中有条件地被激活，以评估血管完整性的功能。体内试验将检测Notch是否有助于或干扰血管内皮生长因子或成纤维细胞生长因子诱导的血管生成。最后，将研究Notch在卵巢中的功能，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。利用荷尔蒙诱导卵巢血管生成的垂体切除小鼠模型，我们将探讨Notch在卵巢血管系统中的作用。这些小鼠将被用来确定当Notch在卵巢血管中有条件地被激活或被使用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过程中的功能。

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.