ILK SIGNAL & CYCLIN DL EXPRESSION POST ARTERIAL INJURY

描述（由申请人提供）：我们最近观察到， 整合素相关信号通路（ILK）在内膜平滑肌细胞的复制中是活跃的， 球囊导管损伤动脉的肌细胞。本提案的重点 是为了确定这个信号通路的激活是否是导致 cyclin D1的表达，并最终用于内膜SMC的复制。 实验设计用于检测ILK下游靶点， 大鼠动脉，并将包括GSK-3的激活，表达和 f3-catenin的核定位和Lef/B-catenin位点的激活 细胞周期蛋白D1启动子。下一个目标将确定上游监管机构， ILK和实验被提议调查整合素的重要性 激活和P13 K通路。封闭抗体将用于抑制B1 和B3整联蛋白活化，并测量ILK活性。 Wortmannin将用于抑制内膜SMC复制中的P13 K通路 和ILK活性定量。最后一个目标将使用带有ILK基因的小鼠 两侧有Lox位点，并将它们与表达平滑肌特异性 克雷老鼠。由此产生的后代将在SMC中缺失ILK，并且这些小鼠 将用于损伤研究。细胞周期蛋白D1表达和SMC复制 然后在颈动脉损伤后的不同时间测量。 策略提出规避的可能性，这样的小鼠不是 可行的最后，表达显性阴性ILK的腺病毒将用于 抑制内膜SMC和细胞周期蛋白D1表达及内膜SMC复制 测定了这些研究将使我们能够证明 细胞周期蛋白D1表达和细胞复制的ILK信号通路 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.