Notch in Angiogenesis and Vascular Biology

阐明控制血管生成的信号通路允许设计用于治疗的新疗法 包括失明、炎症和癌症在内的血管疾病。Notch1 信号传导通常充当血管生成发芽的负调节剂。我们假设Notch 4，一个Notch 主要在内皮细胞中表达的Notch 1受体与Notch 1的作用重叠， 功能协调发展的我们证明Notch 4促进血管生成和可能的血管炎症， 周围的细胞。通过去除小鼠血管中的Notch 4表达或使用抑制剂阻断Notch 4， 我们发现Notch 4在发育中的视网膜中促进血管生成。Notch 4在血管中表达 我们证明，当Notch 4被阻断时，Notch 4抑制肿瘤生长。 肿瘤血管我们发现，内皮Notch 4的功能似乎是改变血管和邻近的基质， 组织.对Notch 4调控基因的分析使我们发现，特定的细胞因子信号蛋白是 受Notch 4而不是Notch 1调节，这是Notch 4对两种血管和 周围组织在这些研究中，我们将定义Notch 4调控的独特信号传导机制， 了解Notch 4如何促进发育，炎症驱动和肿瘤血管生成，并评估 抗Notch 4治疗以获得治疗益处。我们将使用一种新的技术，称为切割和运行，看看哪些基因， 被Notch 4或Notch 1直接激活，并使用新的RiboFlag翻译分析来理解 每个Notch蛋白的表达控制。我们将确定新发现的Notch 4调节蛋白 促进培养的内皮细胞中的血管生成和炎症，并从内皮细胞中去除Notch 4表达。 内皮细胞，以确定这是否会导致血管生长减少， 炎症并减少肿瘤生长。我们将分别从内皮细胞中去除Notch 1或Notch 4基因， 或去除两种基因并描述Notch缺失对发育和病理性血管生成的结果， 为这些基因建立了独特和重叠的作用。一种新的Notch抑制剂使我们能够研究前- 用于治疗增殖性视网膜病（一种炎性眼病）和肿瘤的临床治疗潜力 在内皮细胞中有Notch 4。我们将研究由遗传因素引起的肿瘤间质变化， 或使用流式细胞术和聚焦单细胞RNA测序对Notch 4进行药理学阻断。在所有 通过这些研究，我们将确定在健康和疾病相关的细胞中，选择的细胞因子是否受到Notch 4的调节。 血管，目的是了解Notch 4影响血液炎症的机制 血管和周围组织或肿瘤。我们提出这项建议的目的是要找出 Notch 4可构建正常血管，并有助于病变血管，并评估治疗效果。 预防过度血管生成导致的失明、减少血管炎症和阻断肿瘤的策略。

Elucidating the signaling pathways that control angiogenesis allows new therapies to be designed for treatment of diseases where blood vessels are contributors, including blindness, inflammation, and cancer. Notch1 signaling generally acts as a negative regulator of angiogenic sprouting. We hypothesize that Notch4, a Notch receptor primarily expressed in endothelium, has both overlapping roles with Notch1 and distinct endothelial functions. We demonstrated that Notch4 promotes angiogenesis and possibly inflammation of blood vessels and surrounding cells. By removing Notch4 expression in blood vessels of mice or blocking Notch4 using an inhibitor, we discovered that Notch4 promotes angiogenesis in the developing retina. Notch4 is expressed in blood vessels of many types of tumors and we demonstrated that blocking Notch4 inhibits tumor growth when Notch4 is in tumor vessels. We found that endothelial Notch4 appears to function to alter both vessels and adjacent stromal tissue. Analysis of Notch4-regulated genes led us to discover that specific cytokine signaling proteins are regulated by Notch4, and not Notch1, a possible mechanism for Notch4 broad influence on both vessels and surrounding tissue. In these studies, we will define the unique signaling mechanisms regulated by Notch4 to understand how Notch4 promotes developmental, inflammatory-driven, and tumor angiogenesis and evaluate anti-Notch4 treatment for therapeutic benefit. We will use a new technique called CUT&RUN to see which genes are directly activated by Notch4 or Notch1 and use a novel RiboFlag translational analysis to understand expression control by each Notch protein. We will determine if newly discovered Notch4-regulated proteins promote angiogenesis and inflammation in cultured endothelial cells and remove Notch4 expression from endothelial cells in mice to determine if this causes reduced blood vessel growth, reduces the response to inflammation and reduces tumor growth. We will separately remove Notch1 or Notch4 genes from endothelium or remove both genes and describe result of Notch loss on developmental and pathological angiogenesis, establishing unique and overlapping roles for these genes. A new Notch inhibitor allows us to investigate pre- clinical therapeutic potential for treatment of proliferative retinopathy, an inflammatory eye disease, and in tumors that have Notch4 in the endothelial cells. We will investigate the stromal changes in tumors caused by genetic or pharmacologic blockade of Notch4 using flow cytometry and focused single cell RNA sequencing. In all of these studies, we will determine if select cytokines are regulated by Notch4 in healthy and disease-associated blood vessels, with the goal of understanding the mechanisms by which Notch4 impacts inflammation of blood vessels and surrounding tissue or tumor. Our goals for this proposal will be to discover the unique ways that Notch4 works to build normal blood vessels and contributes to diseased vessels, and to evaluate therapeutic strategies to prevent blindness caused by excess angiogenesis, reduce vascular inflammation, and block tumors.