Prevention of Vein Graft Failure

描述(由申请人提供)：大约1,000,000例主动脉-冠状动脉和外周血管再血管化是使用自体管道进行的。移植物失败的主要原因是随后发生的内膜增生。内膜增生是对损伤的一种反应，涉及到平滑肌的增殖、迁移、表型改变和细胞外基质(ECM)沉积。这项建议将开发一种细胞特指多肽疗法，通过防止导致内膜增生的事件来提高移植物的通畅性。一种小的热休克蛋白HSP27被p38 MAP和MAPKAP激酶II(MK2)的激酶级联反应所磷酸化。磷酸化的HSP27与肌动蛋白应激纤维(肌成纤维细胞表型)的形成和增强的平滑肌迁移有关。我们已经开发出一种抑制MK2的细胞活性多肽。这种多肽还可以抑制应力纤维的形成和细胞外基质的产生。这项建议的具体目的是：具体目标#1：确定抑制HSP27磷酸化的转导多肽对平滑肌生理、形态和生化的影响：我们将确定新型MK2抑制物肽对完整的人血管平滑肌节段和培养的血管平滑肌细胞的影响。特定目的#2：确定优化的多肽模拟物对内膜增生的影响。我们将首先确定MK2抑制剂对人大隐静脉移植器官培养模型中内膜增生的影响。随后，我们将在兔颈动脉插管模型中确定该模拟物的体内效果。具体目标#3：确定磷酸化HSP27“稳定”肌动蛋白细胞骨架的分子机制：我们将使用定量、高通量质谱学技术来分析磷酸化和非磷酸化HSP27的分子关联。该项目的目标是设计生物分子，通过蛋白质转导结构域将多肽直接引入血管细胞，从而提高移植物的通畅性。自体导管是这种治疗方法的理想靶点，因为移植物可以在体外进行治疗，从而为工程蛋白/肽治疗药物的输送提供了最佳的环境。该方案中设计的分子代表了新的疗法，因为药物开发的常见靶点(细胞表面受体和信号级联)被“绕过”，通过改变下游靶标效应蛋白(HSP27)的磷酸化来改变蛋白质-蛋白质的相互作用。

DESCRIPTION (provided by applicant): Approximately 1,000,000 aortocoronary and peripheral vascular revascularizations are performed using autologous conduits. The leading cause of graft failure is the subsequent development of intimal hyperplasia. Intimal hyperplasia represents a response to injury that involves smooth muscle proliferation, migration, phenotypic modulation, and extracellular matrix (ECM) deposition. This proposal will develop a cell permeant peptide therapeutic to enhance graft patency by preventing the events that lead to intimal hyperplasia. A small heat shock protein, HSP27, is phosphorylated by a kinase cascade involving p38 map kinase and MAPKAP kinase II (MK2). Phosphorylated HSP27 is associated with the formation of actin stress fibers (myofibroblast phenotype) and enhanced smooth muscle migration. We have developed a cell permeant peptide that inhibits MK2. This peptide also inhibits stress fiber formation and ECM production. The specific aims of this proposal are: Specific aim #1: Determine the effect of transducible peptides which inhibit the phosphorylation of HSP27 on smooth muscle physiology, morphology, and biochemistry: We will determine the effect of the novel MK2 inhibitor peptide on intact human vascular smooth muscle segments and cultured vascular smooth muscle cells. Specific aim #2: Determine the effect of optimized peptide mimetics on intimal hyperplasia. We will first determine the effect of the MK2 inhibitor on intimal hyperplasia in a human saphenous vein graft organ culture model. Subsequently, we will determine the effect of the mimetics in vivo in a rabbit carotid interposition model. Specific aim #3: Determine the molecular mechanisms by which phosphorylated HSP27 "stabilizes" the actin cytoskeleton: We will use quantitative, high throughput mass spectrometry techniques to analyze the molecular associations of phosphorylated and nonphosphorylated HSP27. The goal of this project is to engineer biomolecules that enhance graft patency using protein transduction domains to directly introduce peptides into smooth muscle cells. Autologous conduits represent an ideal target for this therapeutic approach in that the graft can be treated ex vivo, thus providing an optimal environment for the delivery of engineered protein/peptide therapeutics. The molecules designed in this proposal represent novel therapeutics in that the usual targets of drug development (cell surface receptors and signaling cascades) are "bypassed" and protein-protein interactions are stoichiometrically altered by changing the phosphorylation of downstream target effector proteins (HSP27).