Notch in Angiogenesis and Vascular Biology

阐明控制血管生成的信号通路使新的治疗方法得以设计 与血管有关的疾病，包括失明、炎症和癌症。编号1 信号通常作为血管生成萌发的负调节因子。我们假设Notch4，一种凹槽 受体主要在内皮细胞表达，与Notch1和不同的内皮细胞具有重叠的作用 功能。我们证明了Notch4促进血管生成，并可能促进血管炎症和 周围的细胞。通过去除小鼠血管中Notch4的表达或使用抑制剂阻断Notch4， 我们发现Notch4促进了发育中的视网膜的血管生成。Notch4在血管中表达 在许多类型的肿瘤中，我们证明了当Notch4处于 肿瘤血管。我们发现内皮Notch4似乎具有改变血管和邻近间质的功能 组织。对Notch4调节基因的分析使我们发现特定的细胞因子信号蛋白是 受Notch4而不是Notch1调节，这是Notch4对血管和血管产生广泛影响的可能机制 周围的组织。在这些研究中，我们将定义Notch4调控的独特信号机制 了解Notch4如何促进发育、炎症驱动和肿瘤血管生成，并评估 抗Notch4治疗对治疗有益。我们将使用一种名为Cut&Run的新技术来查看哪些基因 由Notch4或Notch1直接激活，并使用新的RiboFlag翻译分析来理解 表达受每一种Notch蛋白的调控。我们将确定新发现的Notch4调节蛋白 促进培养内皮细胞的血管生成和炎症，并去除Notch4的表达 在小鼠内皮细胞中确定这是否会导致血管生长减少，降低对 发炎，减少肿瘤生长。我们将分别从内皮细胞中移除Notch1或Notch4基因 或者移除这两个基因并描述Notch丢失对发育和病理性血管生成的影响， 为这些基因建立独特和重叠的角色。一种新的Notch抑制剂使我们能够研究 增殖性视网膜病变、炎症性眼病和肿瘤的临床治疗潜力 在血管内皮细胞中有Notch4。我们将研究由遗传基因引起的肿瘤间质变化 或用流式细胞仪和聚焦单细胞RNA测序的方法对Notch4进行药物阻断。在所有的 在这些研究中，我们将确定选定的细胞因子是否受Notch4在健康和疾病相关人群中的调节 血管，目的是了解Notch4影响血液炎症的机制 血管和周围组织或肿瘤。我们这项提案的目标将是发现独特的方式 Notch4的工作是建立正常的血管，促进病变的血管，并评估治疗 预防过度血管生成所致失明、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.