PARACRINE IGF/IGFBP INTERACTIONS IN VIVO

描述：(改编自申请人的摘要)：这是一辆新的R01 Grant专注于使用组织特异性转基因表达来 探讨胰岛素样生长因子-1、胰岛素样生长因子结合蛋白-4和胰岛素样生长因子结合蛋白-5在血管内皮细胞生长和生长中的作用 鼠标中的功能。在初步工作中，调查人员表示， 使用平滑肌α-肌动蛋白启动子(SMP8)的IGF-1和IGFBP-4 允许在子宫、膀胱、肠道和 小动脉。IGF-1表达的表型与 不同组织中器官重塑的不同模式。因此，在 膀胱和胃有向心性增厚的肌层， 而在肠道和子宫中，主要是纵向生长。 IGFBP-4的表达与Smooth发育不全呈对等表型 膀胱、胃和小动脉壁的肌肉。 目前的提案建立在这些初步数据的基础上，包括四个具体的 目标。费金博士将确定IGF-1过度表达对 血管内皮细胞增殖、凋亡和分化的实验研究 这个转基因模型。除了检查细胞的形态外， 组织、细胞有丝分裂比率(通过BUDR标记)和凋亡率 采用TUNEL法进行检测。他还将检查影响 关于收缩能力和对去极化的敏感性。因为他已经 先前证明IGF-1在小鼠模型中过度表达 平滑肌循环中的IGF水平在正常范围内，这将 能够检查这种生长因子在旁分泌的作用 水平。 他将确定IGF-1对血管收缩特性的影响 血管平滑肌。这将包括对钙离子通量的研究。 肌肉，以及血液动力学参数的测量，如血液 压力、外周阻力和脉搏频率。 由于IGFBP-4转基因小鼠发育中等程度的平滑肌 ，他将通过以下方式确定IGFBP的蛋白分解作用 制备携带修饰的IGFBP-4的转基因小鼠 以蛋白酶消化(通过体外诱变)。他还将 联合检测IGF-1和IGFBP-4转基因基因之间的相互作用 表达，因为有数据表明IGF-1与IGFBP-4结合 是蛋白质分解所必需的。 最终的具体目标是，费金博士将使用以下方法确定IGFBP-5的作用 转基因方法。与IGFBP-4相反，IGFBP-5被认为是 与细胞外基质相关，在那里它被稳定和保护 通过蛋白质分解。它还被证明对IGF有抑制作用。 在几种单元格类型中执行操作。为了测试其在体内的生理作用 这种蛋白，研究人员将在他的转基因上表达IGFBP-5 发起人以其原始形式或以在 体外诱变修饰细胞外基质结合部位。他 还将研究将这些小鼠进行IGF转基因的效果 研究共表达对小鼠表型的影响。

DESCRIPTION: (Adapted from the applicant's abstract): This is a new R01 grant which focuses on the use of tissue specific transgenic expression to explore the role of IGF-1 and IGFBP-4 and 5 on smooth muscle growth and function in the mouse. In preliminary work, the investigator has expressed IGF-1 and IGFBP-4 using a smooth muscle a-actin promoter (SMP8) which allows for expression in uterus, urinary bladder, intestines, and arteriole. The phenotype of the IGF-1 expression is quite interesting with different patterns of organ remodeling in different tissues. Thus, in bladder and stomach there is concentric thickening of the muscular layer, whereas in intestine and uterus, there is primarily longitudinal growth. Expression of IGFBP-4 has a reciprocal phenotype with hypoplasia of smooth muscle in bladder, stomach, and arteriole wall. The current proposal builds on these preliminary data with four specific aims. Dr. Fagin will determine the impact of IGF-1 over-expression on smooth muscle proliferation, apoptosis and differentiation in vivo using this transgenic model. In addition to examining the morphology of the tissues, rates of cell mitosis (by BUDR labeling) and rates of apoptosis using the TUNEL assay will be measured. He will also examine the effects on contractility and sensitivity to depolarization. Since he has previously demonstrated that the mouse model of IGF-1 over-expression in smooth muscle has circulating levels of IGF in the normal range, this will enable examination of the action of this growth factor at the paracrine level. He will determine the effects of IGF-1 on contractile properties of vascular smooth muscle. This will include studies of calcium flux in muscles, as well a measurements of hemodynamic parameters such as blood pressure, peripheral resistance and pulse rate. Since IGFBP-4 transgenic mice develop moderate degree of smooth muscle hypoplasia, he will determine the role of proteolysis of IGFBP by generating transgenic mice carrying a modified IGFBP-4 which is resistant to protease digestion (through in vitro mutagenesis). He will also determine the interaction between IGF-1 and IGFBP-4 transgenics by co- expression, since there are data to suggest that IGF-1 binding to IGFBP-4 is required for proteolysis. The final specific aim, Dr. Fagin will determine the role of IGFBP-5 using the transgenic approach. In contrast to IGFBP-4, IGFBP-5 is believed to associated with extra-cellular matrix where it is stabilized and protected from proteolysis. It has also been shown to have inhibitory effect on IGF action in several cell types. To test the in vivo physiological role of this protein, the investigator will express IGFBP-5 on his transgenic promoter either in its native form or in a form which has undergone in vitro mutagenesis to modify the site of extracellular matrix binding. He will also study the effects of crossing these mice will the IGF transgenic mice to study the impact of co-expression on phenotype.