Site-specific therapies to prevent intimal hyperplasia

描述（由申请方提供）：在该提案中，我们假设抗体介导的抗血栓形成/抗炎效应分子靶向血管壁损伤部位可抑制凝血酶和嘌呤能依赖性途径，并增强炎症反应的消退。在此过程中，功能完整的内皮的重建将被加速，再狭窄被抑制。具体而言，我们打算：（1）确定抗体导向的融合蛋白消除导致受损血管壁愈合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和血栓调节蛋白（TM）或直接Xa抑制剂蜱抗凝肽（TAP）的重组抗血栓融合蛋白选择性靶向活化血小板并限制体外和体内促凝血反应的能力。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通过直接作用于中膜SMC或通过改变骨髓源性祖细胞向损伤部位的募集和分化来减少ApoE-/-小鼠主动脉血管成形术模型中内膜增生的程度。(2)确定靶向血管壁损伤部位的CD 39/CD 73在限制导致血栓形成和再狭窄的嘌呤能介导的炎症通路中的有效性。实验将试图确定基于scFv抗LIBS和CD 39和/或CD 73的重组抗血栓形成融合蛋白在体外选择性靶向活化的血小板并介导嘌呤能途径阻断的能力。这些结果将与体内血栓形成、全身抗凝作用的存在和血栓靶向的特异性相关。随后的研究将确定该策略单独或与TM/TAP融合蛋白组合在体内限制血栓生长和内膜增生的有效性。将根据实验结果确定该策略对骨髓源性祖细胞的动员、募集和分化的影响。(3)表征促进损伤诱导的炎症事件消退的生物活性脂质的位点特异性递送用于限制内膜增生的程度。这些研究的第一阶段将寻求确定重组蛋白聚合物胶束靶向体内血管壁损伤部位的能力。在该目标的第二阶段，实验将确定18 R-HEPE、RvE 1和RvD 1的全身与局部递送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的募集和分化方面的作用。最后，我们将评估是否可以进一步优化血管壁愈合时，这两个促血栓形成的过程被废除，并加快炎症事件的解决途径得到促进。 公共卫生相关性：通过使用球囊或激光血管成形术、支架植入术或斑块旋切术治疗下肢血管疾病仍然受到显著再狭窄发生率的限制。我们假设，抑制血管壁损伤后的炎症和血栓形成反应将加速炎症反应的消退和功能完整的内皮的重建。本提案中描述的研究旨在设计抗体导向的抗血栓形成/抗炎效应分子，其靶向血管壁损伤部位，作为抑制内膜增生的策略。

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.