Calcium/ Calmodulin Activated Kinases in Smooth Muscle

项目概述：这个正在进行的研究项目旨在定义多功能 丝氨酸/苏氨酸Ca 2 +/钙调蛋白依赖性蛋白激酶II（CaMKII）同工酶在损伤和疾病中的作用 诱导血管重塑。CaMKII结构复杂，表达为一个大的异源多聚体， 全酶具有12-14个单独的激酶亚基。CAMK 2基因经历广泛的选择性剪接， 产生可以影响全酶亚细胞定位和蛋白质相互作用的变体。中央 假设CaMK II同种型和剪接变体细胞特异性表达和活化是功能性的 损伤和疾病后血管重塑的决定因素。 第一个具体目标将我们在血管平滑肌中CaMKII结构和功能方面的专业知识扩展到 血管内皮，在模拟管腔内动脉损伤的体内小鼠模型的情况下， 支架内再狭窄方面。血管内皮细胞CaMKII亚型在再灌注损伤中的作用 通过条件性敲除特定的Camk 2基因来测试管腔内损伤后的内皮化。 基因工程小鼠STAT 3对CaMKII亚型的调节及CaMKII亚型在细胞凋亡中的作用 在这种炎症环境中测试介导STAT 3依赖性功能。第二个具体目标 利用血管平滑肌中CaMKII变体表达的知识，并测试功能性 CaMKII激酶C末端可变剪接氨基酸序列在介导细胞凋亡形成中的意义 CaMKII κ B/Fyn复合物，以及随后参与血管平滑肌的细胞过程的调节 细胞运动和增殖一种新的CaMKII反义外显子跳跃寡核苷酸（ESO）方法， 设计用于精确干扰该序列的表达的方法已经在体外得到验证。ESO将是 在管腔内动脉损伤之前给予小鼠，目的是限制血管平滑肌 迁移至内膜和随后的新生内膜增生，而不影响血管保护性内皮细胞 再生最后的目的是研究CaMKII亚型在促进程序性细胞死亡中的功能 通过血管平滑肌的坏死性凋亡，这种机制的作用是促进 腹主动脉瘤这一目标是与奥古斯塔大学的实验室合作实现的， 威斯康星州的研究，并使用小鼠模型的主动脉瘤和条件敲除 血管平滑肌中的特异性Camk 2基因。 相关性：实现这些目标将提供关于特定功能重要性的详细信息。 血管损伤、支架再狭窄和血管系统疾病中的CaMKII β亚型变体的应用 靶向CaMKII β的反义ESO可能提供新的治疗方法，以减轻 血管疾病

Project Summary: This ongoing research project is aimed at defining functions of multi-functional serine/threonine Ca2+/Calmodulin-dependent protein kinase II (CaMKII) isozymes in injury- and disease- induced vascular remodeling. CaMKII is structurally complex and is expressed as a large hetero-multimeric holoenzyme with 12-14 individual kinase subunits. CAMK2 genes undergo extensive alternative splicing to produce variants that can affect holoenzyme subcellular localization and protein interactions. The central hypothesis is that cell-specific expression and activation of CaMKII isoforms and splice variants are functional determinants of integrated vascular remodeling in response to injury and disease. The first specific aim extends our expertise in CaMKII structure and function in vascular smooth muscle to the vascular endothelium, in the context of an in vivo mouse model of intraluminal arterial injury that simulates aspects of in-stent restenosis. The functions of vascular endothelial cell CaMKII isoforms in re- endothelialization following intraluminal injury are tested by conditional knockout of specific Camk2 genes in genetically engineered mice. Regulation of the CaMKII isoform by STAT3 and the role of CaMKII in mediating STAT3-dependent functions are tested in this inflammatory environment. The second specific aim capitalizes on knowledge of CaMKII variant expression in vascular smooth muscle and tests the functional significance of an alternatively spliced c-terminal amino acid sequence in CaMKIIthat mediates formation of a CaMKII/Fyn complex, and subsequent regulation of cellular processes involved in vascular smooth muscle cell motility and proliferation A novel CaMKII antisense exon-skipping oligonucleotide (ESO) approach, designed to precisely interfere with expression of this sequence has been validated in vitro. ESOs will be administered to mice prior to intraluminal arterial injury with the goal of limiting vascular smooth muscle migration to the intima and subsequent neointimal hyperplasia, without affecting vasculoprotective endothelial regeneration. The final aim investigates the function of CaMKII isoforms in promoting programmed cell death by necroptosis in vascular smooth muscle and the role of this mechanism is promoting progression of abdominal aortic aneurysm. This aim is carried out in collaboration with labs at Augusta University and the University of Wisconsin and is investigated using mouse models of aortic aneurysm and conditional knockout of specific Camk2 genes in vascular smooth muscle. Relevance: Accomplishing these aims will provide detailed information on the functional importance of specific CaMKII isoform variants in vascular injury, in stent-restenosis and aneurysmal disease. Application of antisense ESOs targeting CaMKII may provide novel therapeutic approaches to mitigate progression of vascular disease.