Site-specific therapies to prevent intimal hyperplasia

描述（由申请人提供）：在本提案中，我们假设抗体介导的抗血栓/抗炎效应分子靶向血管壁损伤部位可以抑制凝血酶和嘌呤能依赖途径，增强炎症反应的消退。在此过程中，功能完整的内皮细胞的重建将被加速，再狭窄将被抑制。具体来说，我们打算：(1)确定抗体导向融合蛋白消除导致血管壁愈合受损的凝血酶依赖性炎症途径的能力。在这些研究的第一阶段，我们计划确定基于scFvanti-LIBS和血栓调节素（TM）或直接Xa抑制剂，tick抗凝肽（TAP）的重组抗血栓融合蛋白在体外和体内选择性靶向活化血小板和限制促凝反应的能力。这些研究的第二阶段将确定这些结构的药代动力学特征，以及全身抗凝作用。在最后阶段，我们将评估表面靶向TM和/或TAP在ApoE-/-主动脉血管成形术小鼠模型中通过直接影响内侧SMCs或通过改变损伤部位骨髓来源祖细胞的募集和分化来减少内膜增生的程度。(2)确定靶向血管壁损伤部位的CD39/CD73在限制嘌呤能介导的导致血栓形成和再狭窄的炎症途径中的有效性。实验将试图确定基于scFvanti-LIBS和CD39和/或CD73的重组抗血栓融合蛋白在体外选择性靶向活化血小板和介导嘌呤能通路阻断的能力。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的特异性相关。后续研究将确定该策略单独使用或与TM/TAP融合蛋白联合使用在体内限制血栓生长和内膜增生的有效性。这一策略对骨髓祖细胞的动员、募集和分化的影响将根据实验结果确定。(3)描述促进损伤性炎症事件解决的生物活性脂质的部位特异性递送在多大程度上限制了内膜增生。这些研究的第一阶段将试图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。在该目标的第二阶段，实验将确定18R-HEPE、RvE1和RvD1在体内限制内膜增生和加速内皮再生方面的相对有效性，以及这些介质（如果有的话）在改变骨髓源性祖细胞募集和分化方面的作用。最后，我们将评估当促血栓形成过程被废除，加速炎症事件解决的途径被促进时，血管壁愈合是否可以进一步优化。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The treatment of lower extremity vascular disease through the use of balloon or laser angioplasty, stenting, or atherectomy remains limited by a significant incidence of restenosis. We hypothesize that inhibiting inflammatory and thrombotic responses after vascular wall injury will accelerate both the resolution of the inflammatory response and the reconstitution a functionally intact endothelium. The investigations described in this proposal are directed at the design of antibody directed anti-thrombotic/anti-inflammatory effector molecules that are targeted to sites of vessel wall injury as a strategy to inhibit intimal hyperplasia.