(Verbatim from the application): Atherosclerosis is a complex disease process characterized by uncontrolled proliferation of vascular smooth muscle cells (vSMCs) and alterations in cell/cell and cell/matrix interactions. Using a chemical model of oxidative injury to the blood vessel wall in vivo, we have recently shown that modulation of vSMCs to proliferative phenotypes involves overexpression of osteopontin (OPN), an integrin ligand, and alterations in integrin-coupled mitogenic signaling. Based on these observations we hypothesized that transition of vSMCs to proliferative phenotypes is mediated by alterations in OPN expression and extracellular matrix (ECM) signaling. To test this hypothesis, studies are proposed in Aim l to define the molecular bases of OPN upregulation as a function of atherogenic status in vSMCs induced by oxidative stress. Nuclear run-on, promoter mutation/deletion analysis, electrophoretic mobility shift assays, mRNA stability measurements, and DNA footprinting will be completed to define the transcriptional and post-transcriptional component of the OPN response. Primary emphasis will be given to NF-KB- and AP-1 coupled signal transduction cascades in the regulation of ECM-regulated gene expression. As part of the second aim, studies will be conducted to characterize the integrin profiles of modified SMCs relative to control counterparts, and to define the molecular basis of signaling interference. Fluorescence activated cell sorting, northern analysis, reverse transcription polymerase chain reaction (PCR), and in situ hybridization will be conducted to identify specific integrins targeted by oxidative injury. The final series of experiments will characterize the interaction between OPN and EGF signaling pathways as a function of phenotypic status.
This aim will involve study of EGF receptor phosphorylation, focal adhesion kinase measurements, and downstream EGF signal transduction cascades to identify signals that lead to convergence of the EGF and ECM signaling during atherogenesis. Collectively, the proposed studies will serve to elucidate molecular mechanisms of integrin-related signaling during the induction of atherogenic vascular SMC phenotypes by oxidative injury. These studies are important in view of the increasing scrutiny of the role of oxidative injury in the onset and progression of atherogenesis.
