Prevention of Vein Graft Failure

描述（由申请人提供）：使用自体导管进行了约1，000，000例冠状动脉和外周血管血运重建。移植物失败的主要原因是内膜增生的后续发展。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。该提案将开发一种细胞渗透肽治疗剂，通过预防导致内膜增生的事件来增强移植物通畅性。一种小的热休克蛋白HSP 27通过涉及p38 map激酶和MAPKAP激酶II（MK2）的激酶级联被磷酸化。磷酸化HSP 27与肌动蛋白应力纤维（肌成纤维细胞表型）的形成和增强平滑肌迁移有关。我们已经开发了抑制MK2的细胞渗透肽。该肽还抑制应力纤维形成和ECM产生。具体目标#1：确定抑制HSP 27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MK 2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。我们将首先在人隐静脉移植器官培养模型中确定MK2抑制剂对内膜增生的影响。随后，我们将在兔颈动脉间置模型中确定模拟物的体内效果。具体目标3：确定磷酸化HSP 27“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HSP 27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，以增强移植物的通畅性。自体导管代表了这种治疗方法的理想靶点，因为移植物可以离体处理，从而为工程蛋白/肽治疗剂的递送提供了最佳环境。在该提案中设计的分子代表了新的治疗方法，因为药物开发的通常靶点（细胞表面受体和信号级联）被“绕过”，并且通过改变下游靶效应蛋白（HSP 27）的磷酸化来化学计量地改变蛋白质-蛋白质相互作用。

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).