ENDOSTATIN RECEPTOR CDNA CLONING AND IONIC SIGNALING

内皮抑素受体 CDNA 克隆和离子信号传导

• 批准号: 6131606
• 负责人: SETH Leo ALPER
• 金额: $ 17.4万
• 依托单位: BETH ISRAEL DEACONESS MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2002-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6131606
• 关键词: Xenopus; Xenopus oocyte; acidity /alkalinity; angiogenesis inhibitors; angiostatins; biological signal transduction; cAMP response element binding protein; calcium flux; collagen; complementary DNA; mechanical stress; molecular cloning; phosphoproteins; receptor; reporter genes; transcription factor; vascular endothelium; voltage /patch clamp

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.

描述：（申请人的描述） 后生动物循环系统通过以下过程经历发育和重塑： 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增殖以及肿瘤生长。 增强血管生成和血管发生是当前的目标 治疗缺血综合征。 相反，抑制血管生成， 是开发新的癌症辅助疗法的当前目标 用于过度增殖的炎症或其他良性疾病。 福克曼及其同事首先假设并证实了新的必要性 毛细血管和微血管生长，使肿瘤持续生长超过临界值， 通常是非致命的，质量由营养物和氧气的供应决定，并且 去除代谢废物。 在众多物质中，Folkman 和 其他随后被鉴定为血管生成的激活剂和抑制剂 已经是具有其他功能的蛋白质的蛋白水解片段。 对象 由于其功效和无毒性，最近备受关注 血管抑制素，促凝血剂，纤维蛋白原的羧基末端片段， 内皮抑素，XVIII 型胶原蛋白的羧基末端片段。 两人都是 最初被鉴定为人类和鼠类小鼠肿瘤生长的抑制剂， 随后对这些分子的生物学研究主要集中在 关于它们对模型系统中血管生成的影响，以及它们对 组织培养细胞中的增殖、细胞进展和凋亡。 其他实验描述了候选蛋白水解途径 它们的生物合成以及最近的结构测定已被 发表。 血管抑制素和血管生成抑制素发挥作用的分子机制仍鲜有研究。 内皮抑素与内皮细胞（可能还有其他靶细胞）相互作用和/或 矩阵，以及由这些触发的假设信号级联 假定的结合相互作用。 细胞内离子作为重要的细胞第二信使 多种细胞类型和信号通路的调节剂。 我们有 因此采用这种方法来研究血管生成抑制剂，并且 发现血管抑制素和内皮抑素都会引发急性 Ca2+ 来自两个大的内皮细胞的原代培养物中的瞬变 和小口径容器。 此类瞬态现象减少或不存在 小组非内皮细胞。 更长时间地接触血管抑制素 内皮抑素导致 Ca2+ 瞬变的衰减 血管生成 VEGF 和 FGF-2。 此外，内皮抑素会引发急性内皮细胞损伤 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，其中我们 建议更详细地研究引起的离子信号通路 内皮细胞通过内皮抑素和血管抑素，并用它们表达 克隆内皮细胞表面内皮抑素受体，并且应该时间 允许，也适用于血管抑制素。 我们将通过追求以下具体目标来实现这些目标： 1. 克隆编码内皮细胞内皮细胞受体内皮抑素和内皮抑素受体的cDNA （时间允许的话）血管抑制素，使用由 这些配体触发细胞内 [Ca2+] 升高的能力。 2. 扩展内皮抑素对 Ca2+ 信号传导的机制表征 和（时间允许）血管抑制素，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. 确定内皮抑素 pH 信号传导的分子基础和（时间 允许）血管抑制素。