Abstract 454: siRNA-mediated Selective Targeting of Signaling Proteins Reveals that ANP Differentially Modulates Cyclic GMP Metabolism, VASP Phosphorylation, and Endothelial Tube Formation and Permeability
Cyclic GMP is synthesized in endothelial cells following ANP activation of the particulate guanylate cyclase GC-A, and also after NO-dependent activation of the soluble guanylate cyclase. The relative contributions of ANP- and NO-modulated changes in cGMP-mediated pathways are incompletely understood. We designed duplex siRNA targeting constructs to knock down selected signaling proteins in bovine aortic endothelial cells (BAEC), and explored receptor-mediated changes in cGMP pathways. ANP activation of the GC-A receptor led to an increase in intracellular cGMP content (determined by EIA) that was rapid (<2min); dose-dependent (EC50 1 nM); and robust (600-fold increase; n=3–4; all p values <0.001). By contrast, activation of soluble guanylate cyclase by nitric oxide agonists led only to a weak (<2-fold) transient increase in endothelial cell cGMP. Increases in cGMP lead to phosphorylation of the vasodilator-stimulated phosphoprotein (VASP). ANP markedly stimulated phosphorylation of VASP Ser239, with a time course and dose response that paralleled the cGMP results. We designed duplex siRNA constructs to target GC-A, VASP, and cyclic GMP-dependent protein kinase (PKG). Transfection of BAEC with these siRNA constructs led to a ~90% decrease in target protein abundance compared to control siRNA. siRNA constructs targeting PKG or GC-A completely abrogated VASP Ser239 phosphorylation. siRNA targeting of PKG blocked endothelial tube formation (n=4) in Matrigel assays; control siRNA was without substantive effect. ANP treatment of BAEC led to a dose-dependent significant decrease in permeability measured using 70 kDa FITC-labeled dextran (31+/−5% decrease, p<0.01, n=4). Prior transfection with siRNA against GC-A, PKG or VASP completely abrogated the ANP-induced decrease in endothelial permeability relative to control siRNA. VASP phosphorylation state has been identified as a marker of endothelial dysfunction, but the molecular pathways that modulate VASP responses are less well characterized. The present studies have used siRNA-based approaches to establish a key role for ANP in GC-A mediated VASP phosphorylation; our findings suggest that this pathway represents an important determinant of endothelial permeability and angiogenesis.