ILK SIGNAL & CYCLIN DL EXPRESSION POST ARTERIAL INJURY

描述(由申请人提供)：我们最近观察到 整合素连接的信号通路(ILK)在复制血管内膜平滑中起活跃作用 球囊导管的肌细胞损伤动脉。这项提案的重点是 就是确定这个信号通路的激活是否负责 细胞周期蛋白Dl的表达最终为复制内膜SMC。 实验是为了检测损伤后ILK的下游靶点。 并将包括激活GSK-3、表达和 F3-连环蛋白的核定位和LEF/B-连环蛋白结合位点的激活 细胞周期蛋白Dl启动子。下一个目标将确定的上游监管机构 ILK和实验被用来研究整合素的重要性 激活和P13K通路。封闭抗体将被用来抑制B1 损伤动脉B3整合素活性及ILK活性测定。 Wortmannin将用于抑制复制内膜SMC中的P13K通路 ILK活性定量。最后一个目标是使用带有ilk基因的小鼠。 两侧是Lox位点，并用表达平滑肌特异性的小鼠来培育它们 Cre小鼠。由此产生的子代将在SMC和这些小鼠中删除ilk 将被用于损伤研究。细胞周期蛋白Dl的表达与SMC复制 然后在颈动脉损伤后的不同时间进行测量。 提出了一些策略，以规避这样一种可能性，即这些老鼠不是 可行。最后，表达显性阴性ilk的腺病毒将用于 转染人血管内膜细胞及细胞周期蛋白的表达和复制 量过了。这些研究将使我们能够证明 细胞周期蛋白Dl表达和细胞周期蛋白复制的ILK信号通路 血管内膜SMC。

DESCRIPTION (provided by the applicant): We have recently observed that the integrin-linked signaling pathway (ILK) is active in replicating intimal smooth muscle cells of balloon catheter injured arteries. The focus of this proposal is to determine if the activation of this signaling pathway is responsible for the expression of cyclin Dl and ultimately for the replication of intimal SMC. Experiments are designed to examine the downstream targets of ILK in injured rat arteries and will include the activation of GSK-3, the expression and nuclear location of f3-catenin and the activation of the Lef/B-catenin sites on the cyclin Dl promoter. The next aim will identify the upstream regulators of ILK and experiments are proposed to investigate the importance of integrin activation and the P13K pathway. Blocking antibodies will be used to inhibit B1 and B3 integrin activation in injured arteries and ILK activity measured. Wortmannin will be used to inhibit the P13K pathway in replicating intimal SMC and ILK activity quantitated. The last aim will use mice with the ILK gene flanked by Lox sites and breed them with mice expressing smooth muscle specific Cre mice. The resultant off spring will have ILK deleted in SMC and these mice will be then used for injury studies. Cyclin Dl expression and SMC replication will then be measured at a various times after carotid artery injury. Strategies are proposed to circumvent the possibility that such mice are not viable. Finally an adenovirus expressing dominant negative ILK will be used to transfect intimal SMC and both cyclin Dl expression and intimal SMC replication measured. These studies will allow us to demonstrate the importance of the ILK-signaling pathway for the expression of cyclin Dl and the replication of intimal SMC.