GEOMETRIC MODULATION OF ENDOTHELIAL NITRIC OXIDE SIGNALING

摘要

NO is a short-lived molecule that rapidly diffuses away from its source making direct measurements of its concentration extremely challenging. The flow conditions needed to study the response to physiological shear stresses further reduces the concentration because of convective transport. Our previous experimental and theoretical studies also suggest that, in addition to the relative levels of expression of signaling molecules involved in NO production, their spatial distribution within the cell is a critical determinant of the resulting signaling behavior. We found that expression levels and distribution of endothelial nitric oxide synthase (eNOS) and its major regulatory protein, caveolin-1 (Cav-1) in cultured endothelial cells can be modulated by controlling cell structure. In particular, elongation and alignment induced by culturing the cells on a substrate with oriented microtopography causes a dramatic redistribution of eNOS such that it becomes colocalized with Cav-1 on the cell surface. We believe this represents a phenotype the more closely approximates a normal endothelium in vivo. Furthermore, on some patterns, the surface distribution of Cav-1 and eNOS becomes non-uniform, concentrating in lines corresponding to actin filament bundles aligned with the microgroove pattern. This phenomenon provides an experimental tool to manipulate the spatial relationships between signaling domains that will allow the systematic testing of hypotheses regarding the role of intracellular transport in NO signaling.