Notch in Angiogenesis and Vascular Biology

描述（由申请人提供）：巨噬细胞和周细胞与新生血管生成有关，它们的功能障碍与多种病理状况有关，如糖尿病、慢性炎症性疾病和肿瘤发生。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。我们的实验室研究Notch信号通路在血管发育和血管生成的背景下。我们实验室最近的数据支持Notch1在巨噬细胞中的高度新颖功能：促进巨噬细胞招募和促进内皮吻合，两个血管芽合并形成功能性血管。周细胞缺陷是糖尿病血管视网膜病变的重要组成部分，周细胞在肿瘤血管生成中起重要作用。我们最近的数据支持Notch信号在发芽血管生成过程中周细胞和内皮细胞之间的串扰中的作用。功能缺失分析表明，周细胞的Notch功能对毛细血管和静脉形态发生至关重要。本研究的总体目标是研究Notch在血管周围细胞中的功能，以了解巨噬细胞和周细胞如何调节血管生成。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，以确定巨噬细胞和周细胞中Notch信号调节的血管生成后果。在Aim I中，我们提出了Notch在巨噬细胞中促进和完善新生血管生成的假设，包括促进内皮细胞吻合。为了探索这一假设，我们将通过基因操纵小鼠视网膜巨噬细胞中的Notch信号，并确定其在视网膜生理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交流的关键配体和Notch蛋白，以阐明Notch下游巨噬细胞调节血管生成的途径。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、周细胞/内皮相互作用的建立以及新生血管的周细胞依赖性稳定中起作用的假设。我们将在基因上操纵周细胞中的Notch活性，以评估有条件地去除Jagged1或Notch1，或从ng2阳性周细胞中完全去除Notch CSL信号的后果。血管生成生长前沿的周细胞将在发育中的视网膜和缺血性视网膜中进行评估。此外，小鼠卵巢将作为模型来研究它们在黄体血管生成中的作用。利用内皮细胞/周细胞共培养来跟踪体外血管形成，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。这些研究阐明了依赖于内皮细胞和血管周围细胞之间相互作用的血管生成的新机制，可能是理解和治疗各种人类血管病变的关键。

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.