Role of CCBE1 in lymphangiogenesis

描述（由申请人提供）：CCBE1是最近发现的一种蛋白质，在患有严重遗传性皮肤和肠道肿胀的患者中发生突变，称为“淋巴水肿-淋巴管扩张-智力迟钝综合征”或Hennekam综合征。在这些患者中，淋巴功能紊乱，淋巴是一个中空的盲端管网络，从身体组织中收集血管外液体，并将这些液体返回血液循环。CCBE1如何调节淋巴功能仍然完全未知。斑马鱼是一种小型淡水鱼，与鲦鱼有亲缘关系，ccbe1基因的突变会导致所有淋巴系统缺失，严重肿胀，最终死亡。这些突变的鱼与斑马鱼有着惊人的相似之处，它们都破坏了一种不同的基因，vegf-c，这种基因编码了以前已知的唯一能特异性刺激淋巴生长的因子。因此，CCBE1似乎是第二种能够刺激淋巴生长的蛋白质，与VEGF-C并肩作用。本研究旨在利用转基因小鼠和人类淋巴细胞进一步探索CCBE1在淋巴生长中的作用。这些研究将侧重于确定CCBE1在皮肤中的作用，皮肤是淋巴功能缺陷患者的严重影响组织，特别是在Hennekam综合征中。在这项工作中，我们将首先通过在小鼠的DNA中插入CCBE1基因的额外拷贝来测试CCBE1是否足以刺激淋巴生长。这些额外的基因拷贝将被设计成仅在皮肤中产生额外的CCBE1蛋白。我们将评估这些动物皮肤中的淋巴管，以确定额外的ccbe1蛋白是否能够诱导淋巴管在大小、数量或功能上的变化。其次，我们将我们将产生具有无功能Ccbe1基因的小鼠。我们将使用这些小鼠在所有组织中删除Ccbe1，然后单独删除皮肤，以检查哺乳动物淋巴发育和皮肤中对Ccbe1的需求。最后，我们将在培养皿中测试CCBE1蛋白直接促进淋巴内皮细胞生长和存活的能力。在这个简化的系统中，我们将用从人皮肤淋巴管中分离的细胞培养ccbe1蛋白。我们将仔细测量细胞生长、存活和内部信号的变化。

DESCRIPTION (provided by applicant): CCBE1 is a protein recently discovered to be mutated in patients with a severe inherited form of swelling in their skin and intestines known as "Lymphedema-lymphangiectasia-mental retardation syndrome" or Hennekam Syndrome. In these patients, there is dysfunction of the lymphatics, a network of hollow, blind-end tubes, which collect extravascular fluid from the tissues of the body and return this fluid to the blood circulation. How CCBE1 regulates lymphatic function remains completely unknown. In the zebrafish, a small freshwater fish related to the minnow, mutation of the ccbe1 gene leads to absence of all lymphatics, severe swelling, and ultimately death of the fish. These mutant fish have a striking similarity with zebrafish with disruption of a different gene, vegf-c, which encodes the only factor previously known to specifically stimulate growth of lymphatics. CCBE1, therefore, appears to be a second protein capable of stimulating lymphatic growth, acting side-by-side with VEGF-C. This work seeks to further explore the role of CCBE1 in lymphatic growth using genetically modified mice and cultured cells derived from human lymphatics. These studies will focus on defining the role of CCBE1 in the skin, a tissue severely affected in patients with defects in lymphatic function generally, and in Hennekam Syndrome specifically. In this work, we will first test if CCBE1 alone is sufficient to stimulate lymphatic growth by creating mice that have extra copies of the Ccbe1 gene inserted into their DNA. These additional copies of the gene will be engineered to produce additional CCBE1 protein in the skin only. We will evaluate the lymphatics in the skin of these animals to determine if the additional CCBE1protein is capable of inducing changes in lymphatic in size, number, or function. Secondly, we will we will generate mice with a non-functional Ccbe1 gene. We will use these mice to delete Ccbe1 both in all tissues and then the skin alone, to examine the requirement for CCBE1 in mammalian lymphatic development and in the skin specifically. Finally, we will test the ability of CCBE1 protein to directly promote growth and survival of lymphatic endothelial cells in a culture dish. In this simplified system, we will incubate ccbe1 protein with cells isolated from human skin lymphatic vessels. We will carefully measure changes in cell growth, survival, and internal signaling. PUBLIC HEALTH RELEVANCE: This work will study how CCBE1, a gene mutated in patients with a form of severe swelling of skin and gut, regulates lymphatic growth. We will generate mice with either targeted deletion or increased expression of CCBE1 in the skin. We will also test the effects of the protein on lymphatic endothelial cells in culture.