Notch in Angiogenesis and Vascular Biology

描述(申请人提供)：巨噬细胞和周细胞与血管生成有关，它们的功能障碍与糖尿病、慢性炎症性疾病和肿瘤形成等多种病理情况有关。尽管巨噬细胞与新生血管密切相关，但对其在生理性和病理性血管生成中的作用知之甚少。我们的实验室在血管发育和血管生成的背景下研究了Notch信号通路。我们实验室的最新数据支持Notch1在巨噬细胞中具有非常新的功能：促进巨噬细胞募集和促进内皮细胞吻合，两个血管芽合并形成一个功能性血管。周细胞缺陷是糖尿病视网膜血管病变的重要组成部分，周细胞对肿瘤血管生成具有重要作用。我们最近的数据支持Notch信号在血管萌发过程中周细胞和内皮细胞之间的串扰中所起的作用。功能丧失分析表明，周细胞中的Notch功能对毛细血管和静脉的形态发生至关重要。这项建议的总体目标是研究血管周围细胞的Notch功能，以了解巨噬细胞和周细胞如何调控血管生成。我们的总体策略将结合遗传小鼠模型和互补的体外血管生成试验来确定Notch信号调节在巨噬细胞和周细胞中的血管生成后果。在目标I中，我们继续提出假设，即Notch在巨噬细胞中起促进和细化萌芽血管生成的作用，包括促进内皮细胞的吻合。为了探索这一假说，我们将从基因上操纵小鼠视网膜巨噬细胞中的Notch信号，并确定其在生理性视网膜发育和缺血性视网膜病变模型中对血管生成的贡献。我们将评估可能参与巨噬细胞和内皮细胞之间通讯的关键配体和Notch蛋白，以阐明Notch下游调节血管生成的巨噬细胞途径。在AIM II中，我们提出了体内和体外方法来检验周细胞Notch信号在静脉和毛细血管分化、周细胞招募、建立周细胞/内皮细胞相互作用以及周细胞依赖的新生血管稳定中的作用。我们将从基因上操纵周细胞中的Notch活性，以评估有条件地从NG2阳性的周细胞中移除Jagged1或Notch1，或完全去除Notch CSL信号的后果。处于血管生成生长前沿的周围眼将在发育中和缺血的视网膜中进行评估。此外，还将以小鼠卵巢为模型，研究它们在黄体血管生成中的作用。利用内皮细胞/周细胞共培养跟踪体外血管形成，我们将进一步阐明Notch和Notch配体在内皮细胞和周细胞中的作用。这些研究阐明了血管生成的新机制，这些机制依赖于血管内皮细胞和血管周围细胞之间的相互作用，可能是理解和治疗各种人类血管病理的关键。

DESCRIPTION (provided by applicant): Macrophages and pericytes have been implicated in sprouting angiogenesis, and their dysfunction is linked to diverse pathological conditions such as diabetes, chronic inflammatory disease, and tumorigenesis. Little is known about the role of macrophages in physiological and pathological angiogenesis, despite their close association with newly growing vessels. Our laboratory studies the Notch signaling pathway in the context of vascular development and angiogenesis. Recent data from our lab supports highly novel functions for Notch1 in the macrophage: facilitation of macrophage recruitment and promotion of endothelial anastomosis, the merging of two vascular sprouts to form a functional vessel. Pericyte defects are a prominent component of diabetic vascular retinopathies, and pericytes are important for tumor angiogenesis. Our recent data supports a role for Notch signaling in the crosstalk between pericytes and endothelial cells during sprouting angiogenesis. Loss of function analysis demonstrated that Notch function in pericytes is critical for capillary and vein morphogenesis. The overall objective of this proposal is to study Notch function in peri- vascular cells in order to understand how macrophages and pericytes regulate angiogenesis. Our general strategy will combine genetic mouse modeling and complementary in vitro angiogenesis assays to determine the angiogenic consequences of Notch signaling modulation in macrophages and pericytes. In Aim I, we pursue the hypothesis that Notch functions in macrophages to promote and refine sprouting angiogenesis, including facilitation of endothelial anastomosis. To explore this hypothesis we will genetically manipulate murine Notch signaling in the retinal macrophages, and determine its contribution to angiogenesis in both physiological retinal development and a model of ischemic retinopathy. We will evaluate key ligands and Notch proteins that may participate in communication between macrophages and endothelium, to clarify the macrophage pathways that function downstream of Notch to regulate angiogenesis. In Aim II, we propose in vivo and in vitro approaches to examine the hypothesis that pericytes Notch signaling functions in vein and capillary differentiation, pericyte recruitmen, establishment of pericyte/endothelial interactions, and in pericyte- dependent stabilization of nascent vessels. We will genetically manipulate Notch activity in pericytes to assess the consequences of conditional removal of either Jagged1 or Notch1, or total ablation of Notch CSL signaling, from NG2-positive pericytes. Perictyes at the leading front of angiogenic growth will be evaluated in both developing and ischemic retinas. Additionally, the mouse ovary will be used as a model to study their role in luteal angiogenesis. Using endothelial cell/pericyte co-cultures to follow vessel formation in vitro, we will further clarify the roles of Notch and Notch ligands in endothelial cells versus pericytes. These studies elucidate novel mechanisms of angiogenesis that depend on interactions between endothelial and peri-vascular cells, and may be key to the understanding and treatment of a variety of human vascular pathologies.