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).
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