COBRE: LSU HSC: P3: GROWTH FACTOR & INTEGRIN SIGNALING IN VSMC

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proliferation and migration of vascular smooth muscle cells (VSMC) occur rapidly after vessel injury concomitant with switching of VSMC from a contractile to a synthetic phenotype. These changes in VSMC behavior contribute to intimal expansion and vascular remodeling seen in atherosclerotic plaques. Phenotypic switching is associated with changes in the local extracellular matrix that function to suppress or enhance VSMC proliferation, migration or contractility. The MAP kinase pathway governs each of these endpoints, and is regulated by stimulatory and inhibitory adhesion signals. Three Specific Aims were proposed to dissect how adhesion signals regulate ERK signaling in VSMC. The progress towards completion of Aims I - III made during the second budget period is summarized below: Aims I/II. Determine the matrix-specific modulation of MAP kinase activation by growth factor stimulation in VSMC. We have visualized phospho-ERK in femoral artery sections from balloon catheter-injured rats. Intimal expansion is apparent 3-7 days after injury (3 rats per time point). Furthermore, co-staining with anti phospho-ERK and anti smooth muscle actin antisera reveals that ERK is activated in smooth muscle cells at the injury site at these time points following injury. In collaboration with Dr. Park we have generated lentiviral constructs for the expression of epitope-tagged MEK mutants and other signaling molecules in vascular endothelial and smooth muscle cells in situ. We will express phosphorylation-defective and phosphorylation-mimetic MEK1 in situ to test the hypothesis that MEK1 phosphorylation controls ERK activation and VSMC function during restenosis. Aim III. Identify matrix-specific MAP kinase substrates following growth factor stimulation in VSMC. Initially we proposed to identify and characterize matrix-dependent ERK substrates in VSMC in culture before asking whether these targets are important in vivo. However, difficulties encountered in our tissue culture model have led us to attempt to identify putative ERK substrates regulated in concert with VSMC phenotypic switching in situ in balloon-injured rabbit vessel sections. Our strategy is to use lentiviral reagents to manipulate MEK and ERK signaling in situ, and phosphoprotein-enrichment combined with mass spectrometry to identify putative ERK substrates in vessel tissue. We have made the necessary viral constructs, and "proof of principle" experiments have identified phosphorylation sites in multiple VSMC proteins.