ENDOSTATIN RECEPTOR CDNA CLONING AND IONIC SIGNALING

描述：(申请人描述) 后生动物循环系统经历了发育和重塑 血管生成、血管生成和动脉生成的过程。 血管生成现在被认为是胚胎发育的核心过程， 器官发生和再生组织的增殖与肿瘤的生长。 促进血管生成和血管生成是目前的目标 脑缺血综合征的治疗。相比之下，抑制血管生成， 是开发新的癌症辅助疗法的当前目标 用于炎症性或其他良性疾病的过度增殖。 福克曼和他的同事首先假设并证实了新的 毛细血管和微血管生长使肿瘤持续生长超过临界、 通常不致命，质量由营养和氧气的供应决定，以及 新陈代谢废物的去除。在众多物质中，福克曼和 其他后来被确认为血管生成激活剂和抑制物的化合物 已经是具有其他功能的蛋白质的蛋白水解性片段。的宗旨 由于它们的有效性和无毒性，最近受到了很大的关注 血管抑素，促凝剂纤维蛋白原的羧基末端片段， 内皮抑素，XVIII胶原蛋白的一个羧基末端片段。两人都是 最初被确定为抑制人类和小鼠肿瘤生长的药物， 随后对这些分子的生物学研究主要集中在 在模型系统中对血管生成的影响，以及对血管生成的影响 组织培养细胞的增殖、细胞进展和凋亡。 其他实验，已经描述了候选的蛋白质分解途径 它们的生物合成和最近的结构确定一直是 出版了。 血管抑素和血管紧张素转换酶的分子机制研究较少 内皮抑素与内皮细胞和/或其他靶细胞相互作用 和矩阵，以及由这些触发的假想的信号级联 假定的结合相互作用。 细胞内离子是重要的细胞第二信使和 调节剂存在于多种细胞类型和信号通路中。我们有 因此采用这种方法来研究血管生成抑制剂，并 已经发现血管抑素和内皮抑素都能触发急性钙离子 大鼠血管内皮细胞原代培养中的瞬变 和小口径的舰艇。这样的瞬变会减少或在 非内皮细胞的小面板。更长时间的血管抑素暴露 和内皮抑素导致由血管生成的钙瞬变的减弱 血管生成的血管内皮生长因子和成纤维细胞生长因子-2。此外，内皮抑素还可触发急性内皮细胞 细胞碱化。 这些观察结果构成了这项为期两年的R2I提案的基础，在该提案中，我们 建议更详细地研究在体内诱导的离子信号通路 内皮细胞通过内皮抑素和血管抑素，并利用它们来表达 克隆内皮细胞表面内皮抑素受体，并应适时 许可，血管抑素也是如此。 我们将通过实现以下具体目标来实现这些目标： 1.克隆内皮抑素和内皮细胞受体的编码基因 (时间允许)Angiostatin，使用由 这些配体触发细胞内[Ca2+]升高的能力。 2.内皮抑素在钙信号转导机制中的应用 和(时间允许的)血管抑素，包括与内皮细胞的相互作用 对机械和非渗透扰动的反应。 3.内皮抑素和(Time)在pH信号转导中的分子基础 允许的话)血管抑素。

DESCRIPTION: (Applicant's Description) The metazoan circulatory system undergoes development and remodeling through the processes of vasculogenesis, angiogenesis, and arteriogenesis. Angiogenesis is now recognized as a process central to embryonic development, organogenesis and regenerative tissues proliferation, and tumor growth. Enhancement of angiogenesis and vasculogenesis is a current goal in the treatment of ischemic syndromes. Inhibition of angiogenesis, in contrast, is a current goal in the development of new adjunct therapies for cancer and for inflammatory or other benign disorders of hyperproliferation. Folkman and colleagues First postulated and confirmed the necessity of new capillary and microvessel growth for sustained tumor growth beyond a critical, usually nonlethal, mass determined by supply of nutrients and oxygen, and removal of metabolic waste products. Among the many substances Folkman and others subsequently identified as activators and inhibitors of angiogenesis have been proteolytic fragments of proteins with other functions. Objects of much recent attention due to their efficacy and lack of toxicity have been angiostatin, a carboxy-terminal fragment of the procoagulant, fibrinogen, endostatin, a carboxy-terminal fragment of collagen XVIII. Both were originally identified as inhibitors of human and murine tumor growth in mice, and subsequent biological investigation of these molecules has largely focused on their effects on angiogenesis in model system, and their effect on proliferation, cell progression, and apoptosis in tissue culture cells. Additional experiments, have described candidate proteolytic pathways for their biosynthesis and, more recently, structure determinations have been published. Still little studied has been the molecular mechanism by which angiostatin and endostatin interact with endothelial (and perhaps other target) cells and/or with matrix, and the hypothesized signaling cascades triggered by these putative binding interactions. Intracellular ions serve as important cellular second messengers and modulators in a wide variety of cell types and signaling pathways. We have therefore taken this approach to the study of angiogenesis inhibitors, and have discovered that both angiostatin and endostatin trigger acute Ca2+ transients in primary cultures of endothelial cells derived from both large and small-caliber vessels. Such transients are reduced or absent among a small panel of non-endothelial cells. More prolonged exposure to angiostatin and endostatin leads to attenuation of the Ca2+ transients produced by the angiogenic VEGF and FGF-2. In addition, endostatin triggers acute endothelial cell alkalinization. These observations form the basis of this two-year R2I proposal, in which we propose to study in greater detail the ionic signaling pathways elicited in endothelial cells by endostatin and angiostatin, and to use them to expression clone endothelial cell surface receptors for endostatin and, should time permit, for angiostatin as well. We will accomplish these objectives by pursuit of the following Specific Aims: 1. To clone cDNAs encoding endothelial cell receptors for endostatin and (time permitting) angiostatin, using parallel strategies enabled by the ability of these ligands to trigger elevations in intracellular [Ca2+]. 2. To extend our mechanistic characterization of Ca2+ signaling by endostatin and (time permitting) angiostatin, including interactions with endothelial responses to mechanical and aniosmotic perturbations. 3. To determine the molecular basis of pH-signaling by endostatin and (time permitting) angiostatin.