PARACRINE IGF/IGFBP INTERACTIONS IN VIVO

描述：（改编自申请人摘要）：这是一个新的R 01 该基金专注于利用组织特异性转基因表达， 探索IGF-1和IGFBP-4和5对平滑肌生长的作用， 鼠标中的函数在初步工作中，调查人员表示， 使用平滑肌α-肌动蛋白启动子（SMP 8）的IGF-1和IGFBP-4， 允许在子宫、膀胱、肠中表达， 小动脉IGF-1表达的表型非常有趣， 不同组织的器官重塑模式不同。因此在 膀胱和胃有同心增厚的肌肉层， 而在肠和子宫中，主要是纵向生长。 IGFBP-4的表达与平滑肌细胞发育不全具有相反的表型， 膀胱、胃和小动脉壁中的肌肉。 目前的提案以这些初步数据为基础， 目标。Fagin博士将确定IGF-1过度表达对 平滑肌增殖、凋亡和分化 这个转基因模型。除了检查的形态， 组织、细胞有丝分裂率（通过BUDR标记）和细胞凋亡率 使用TUNEL测定将被测量。他还将研究 收缩性和对去极化的敏感性。既然他 先前证明，IGF-1过表达的小鼠模型， 平滑肌的IGF循环水平在正常范围内， 能够检查这种生长因子在旁分泌中的作用， 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研究钙通量， 肌肉，以及血液动力学参数的测量， 压力、外周阻力和脉搏率。 由于IGFBP-4转基因小鼠发育出中度平滑肌， 发育不全，他将确定IGFBP蛋白水解的作用， 产生携带经修饰的IGFBP-4的转基因小鼠，所述经修饰的IGFBP-4具有抗性， 蛋白酶消化（通过体外诱变）。他还将 确定IGF-1和IGFBP-4转基因之间的相互作用， 由于有数据表明IGF-1与IGFBP-4的结合 是蛋白水解所必需的。 最后的具体目标，Fagin博士将确定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 从蛋白质水解中它也被证明对IGF有抑制作用 在几种细胞类型中起作用。为了测试体内生理作用， 这种蛋白质，研究者将表达IGFBP-5对他的转基因 启动子以其天然形式或以已经经历过转化的形式存在。 体外诱变以修饰细胞外基质结合位点。他 还将研究这些小鼠杂交的影响将IGF转基因 小鼠来研究共表达对表型的影响。

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.