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.
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.
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