Site-specific therapies to prevent intimal hyperplasia

项目概要： 在这个提议中，我们假设抗体介导的抗血栓/抗炎效应物的靶向作用可能是通过抑制抗血栓/抗炎效应物的表达来实现的。 分子的血管壁损伤部位可以抑制凝血酶和嘌呤能依赖性途径，并增强 炎症反应的消退。在这个过程中，功能完整的内皮细胞的重建将是 加速和抑制再狭窄。具体而言，我们打算： (1)确定抗体导向的融合蛋白消除凝血酶依赖性炎症反应的能力。 导致血管壁愈合受损的途径。在这些调查的第一阶段，我们计划 定义基于scFvanti-LIBS和血栓调节蛋白的重组抗血栓形成融合蛋白的能力 (TM)或直接Xa抑制剂，蜱抗凝肽（TAP），以选择性地靶向活化的血小板， 体外和体内的促凝血反应。这些研究的第二阶段将确定药代动力学特征 以及全身抗凝作用。在最后阶段，我们将评估 在ApoE-/-小鼠主动脉血管成形术模型中，表面靶向TM和/或TAP通过直接 影响中膜SMC或通过改变骨髓来源的祖细胞的募集和分化， 受伤的地方。 (2)确定靶向血管壁损伤部位的CD 39/CD 73在限制嘌呤能 介导的炎症途径，导致血栓形成和再狭窄。实验将寻求 为了定义基于scFv抗LIBS和CD 39和/或CD 39的重组抗血栓形成融合蛋白的能力， CD 73选择性靶向活化血小板并介导嘌呤能通路阻断的体外研究这些结果将 与体内血栓形成、全身抗凝作用的存在以及 血栓靶向随后的调查将确定这一战略的有效性，无论是单独还是在 与TM/TAP融合蛋白组合以限制体内血栓生长和内膜增生。这样做的影响 关于骨髓来源的祖细胞的动员、募集和分化的策略将是 这是由实验结果决定的。 (3)表征生物活性脂质的位点特异性递送在多大程度上促进了 损伤诱导的炎症事件用于限制内膜增生。这些研究的第一阶段将寻求 以确定重组蛋白聚合物胶束在体内靶向血管壁损伤部位的能力。在 在这一目标的第二阶段，实验将确定全身与局部递送的相对有效性。 18 R-HEPE、RvE 1和RvD 1在体内限制内膜增生和加速内皮再生中的作用， 这些介质（如果有的话）在改变骨髓来源的祖细胞的募集和分化中的作用。 最后，我们将评估当两个血栓形成前过程同时发生时，血管壁愈合是否可以进一步优化。 消除，并促进加速炎症事件消退的途径。

PROJECT SUMMARY: In this proposal, we hypothesize that antibody mediated targeting of anti-thrombotic/anti-inflammatory effector molecules to sites of vessel wall injury can inhibit thrombin and purinergic dependant pathways and enhance the resolution of the inflammatory response. In the process, the reconstitution of a functionally intact endothelium will be accelerated and restenosis inhibited. Specifically, we intend to: (1) Define the capacity of antibody-directed fusion proteins to abrogate thrombin-dependant inflammatory pathways that contribute to impaired vascular wall healing. In the first phase of these investigations, we plan to define the capacity of recombinant anti-thrombotic fusion proteins based upon scFvanti-LIBS and either thrombomodulin (TM) or the direct Xa inhibitor, tick anticoagulant peptide (TAP), to selectively target activated platelets and limit procoagulant responses in vitro and in vivo. The second phase of these studies will define the pharmacokinetic profile of these constructs, as well as systemic anticoagulant effects. In the final phase, we will evaluate the extent to which surface targeted TM and/or TAP reduce intimal hyperplasia in an ApoE-/- mouse model of aortic angioplasty by a direct effect on medial SMCs or by altering the recruitment and differentiation of bone marrow derived progenitor cells to the site of injury. (2) Determine the effectiveness of CD39/CD73 targeted to sites of vessel wall injury in limiting purinergic mediated inflammatory pathways that contribute to thrombus formation and restenosis. Experiments will seek to define the ability of recombinant anti-thrombotic fusion proteins based upon scFvanti-LIBS and either CD39 and/or CD73 to selectively target activated platelets and mediate purinergic pathway blockade in vitro. These results will be correlated with thrombus formation in vivo, the presence of systemic anticoagulant effects, and the specificity of thrombus targeting. Subsequent investigations will determine the effectiveness of this strategy either alone or in combination with TM/TAP fusion proteins to limit thrombus growth and intimal hyperplasia in vivo. The impact of this strategy on the mobilization, recruitment, and differentiation of bone marrow derived progenitor cells will be determined, as dictated by experimental findings. (3) Characterize the extent to which site-specific delivery of bioactive lipids that promote the resolution of injury induced inflammatory events serve to limit intimal hyperplasia. The first phase of these studies will seek to define the capacity of recombinant protein polymer micelles to be targeted to sites of vessel wall injury in vivo. In the second phase of this aim, experiments will determine the relative effectiveness of systemic versus local delivery of 18R-HEPE, RvE1, and RvD1 in limiting intimal hyperplasia and accelerating endothelial regeneration in vivo and the role of these mediators, if any, on altering the recruitment and differentiation of bone marrow derived progenitor cells. Finally, we will assess whether vascular wall healing can be further optimized when both pro-thrombotic processes are abrogated and pathways that accelerate the resolution of inflammatory events are promoted.