Prevention of Vein Graft Failure

描述（由申请人提供）：使用自体导管进行了大约1,000,000例冠状动脉和周围血管重建术。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反应，涉及平滑肌增殖、迁移、表型调节和细胞外基质（ECM）沉积。本建议将开发一种细胞渗透肽疗法，通过防止导致内膜增生的事件来增强移植物的通畅性。一种小的热休克蛋白，HSP27，通过涉及p38 map激酶和MAPKAP激酶II （MK2）的激酶级联磷酸化。磷酸化的HSP27与肌动蛋白应激纤维（肌成纤维细胞表型）的形成和平滑肌迁移增强有关。我们已经开发出一种抑制MK2的细胞渗透肽。这种肽也抑制应激纤维的形成和ECM的产生。该提案的具体目标是：具体目标#1：确定抑制HSP27磷酸化的可转导肽对平滑肌生理学、形态学和生物化学的影响：我们将确定新型MK2抑制剂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。具体目标2：确定优化的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，我们将在兔颈动脉介入模型中确定模拟物的体内效果。具体目标3：确定磷酸化的HSP27“稳定”肌动蛋白细胞骨架的分子机制：我们将使用定量、高通量质谱技术来分析磷酸化和非磷酸化的HSP27的分子关联。该项目的目标是设计生物分子，利用蛋白质转导结构域直接将肽引入平滑肌细胞，增强移植物的通畅性。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治疗，从而为工程蛋白/肽疗法的递送提供了最佳环境。在这个提议中设计的分子代表了一种新的治疗方法，因为药物开发的通常靶标（细胞表面受体和信号级联）被“绕过”，蛋白质-蛋白质相互作用通过改变下游靶效应蛋白（HSP27）的磷酸化而发生化学计量改变。

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).