The Role of CLIC4 in Angiogenesis

描述（由申请人提供）：血管生成，即现有血管形成新血管的过程，是发育和对环境和疾病反应的关键过程。血管发育和血管生成的改变在许多不同的疾病中起重要作用，包括糖尿病、冠状动脉疾病、中风和癌症。更全面地了解导致适当控制血管生成的机制可能会为预防和/或治疗这些常见的使人衰弱的疾病带来新的方法。毛细血管是由内皮细胞通过“细胞空化”的过程形成的，这个过程包括细胞内囊泡的形成和融合，形成一个大的细胞内液泡，最终成为小管的细胞外管腔。CLIC蛋白是一类主要存在于细胞膜内的氯离子通道蛋白。氯离子的通透性长期以来被认为在细胞膜内运输中起作用，但负责细胞膜内单个氯离子传导的特定蛋白质在很大程度上仍然未知。最近，一种CLIC蛋白已被证明对秀丽隐杆线虫特定的细胞空心化小管形成过程至关重要，而家族成员CLIC4也与哺乳动物内皮细胞的体外小管形成有关。我们最近培育并鉴定了CLIC4基因被破坏的小鼠。来自这些小鼠的初步数据提供了令人震惊和兴奋的新证据，表明CLIC4确实参与血管生成。我们的假设是，CLIC4在血管生成中起着关键作用，可能是通过提供氯离子电导来实现有序的膜传输和参与内皮细胞空化小管形成的膜融合步骤。我们建议：1)研究CLIC4（-/-）小鼠血管发育异常和对血管生成刺激的反应；2)确定CLIC4在细胞空化小管发生中所必需的序列和基序；3)研究源自细胞的膜的运输特性，以研究可归因于CLIC4的活动，并探讨其如何影响管形成。因此，该提案是一个假设驱动的基础科学项目，它使用新的方法和试剂来解决最近确定的血管生成的关键步骤，目前对其机制知之甚少。结果将有明确的临床相关性在许多疾病中，血管生成发挥作用的一部分。公共卫生相关性：新血管的生长和调节对正常发育很重要，对癌症、心脏病和中风等几种重要疾病也至关重要。这一建议的重点是新发现的细胞内氯离子通道在新血管发育中的关键作用。拟议研究的结果将为这些常见的重要疾病过程提供新的见解，并可能为预防和治疗开辟新的途径。

DESCRIPTION (provided by applicant): Angiogenesis, the process that new blood vessels form from existing vessels, is a critical process in development and response to environment and disease. Alterations in vascular development and angiogenesis play important roles in many diverse disease including diabetes, coronary artery disease, stroke, and cancer. More complete understanding of the mechanisms leading to appropriately controlled angiogenesis may lead to new approaches for prevention and/or treatment of these common debilitating diseases. Capillary tubules form from endothelial cells by a process called "cell-hollowing" tubulogenesis, which involves formation and fusion of intracellular vesicles to form a large intracellular vacuole that eventually becomes the extracellular lumen of the tubule. CLIC proteins are a family of chloride channel proteins which largely reside in intracellular membranes. Chloride permeability has long been recognized to play a role in intracellular membrane traffic but the specific proteins responsible for individual chloride conductance of intracellular membranes is still largely unknown. Recently, a CLIC protein has been shown to be essential for a specific cell-hollowing tubulogenic process in C. elegans and the family member CLIC4 has been implicated in tubulogenesis in vitro of mammalian endothelial cells. We have recently generated and characterized mice in which the gene for CLIC4 has been disrupted. Preliminary data from these mice provide startling and exciting new evidence that CLIC4 is indeed involved in angiogenesis. Our hypothesis is that CLIC4 plays a critical role in angiogenesis, perhaps by providing the chloride conductance necessary for the orderly membrane traffic and membrane fusion steps involved in cell-hollowing tubulogenesis of endothelial cells. We propose to: 1) Study CLIC4 (-/-) mice for abnormalities in vascular development and response to angiogenic stimuli; 2) Identify sequences and motifs within CLIC4 that are necessary for its function in cell-hollowing tubulogenesis; and 3) Investigate transport properties of membranes derived from cells undergoing tubulogenesis for an activity attributable to CLIC4 and explore how that effects tubulogenesis. The proposal thus is a hypothesis-driven basis science project that uses novel approaches and reagents to address a recently-identified key step in angiogenesis about which little is currently known mechanistically. The results will have clear clinical relevance to the many diseases in which angiogenesis plays a part. PUBLIC HEALTH RELEVANCE: Growth and regulation of new blood vessels is important in normal development and critical in several important diseases such as cancer, heart disease and stroke. This proposal focuses on newly discovered key role for an intracellular chloride channel in development of new blood vessels. The results of the proposed studies will provide new insight into these common, important disease processes and may open new avenues for prevention and treatment.