Consistent knock down of PKG required maintaining cells in standard culture medium. of the transcription factor ELK-1, and heparin induced MKP-1 synthesis. Although transient, the levels of cGMP increase in heparin treated cells. Finally, knock down of protein kinase G also significantly decreases heparin effects in growth factor activated vascular smooth muscle cells. Together, these data indicate that heparin effects on vascular smooth muscle cell proliferation depend, at least in part, on signaling through protein kinase G. == INTRODUCTION == Following injury, migration of vascular smooth muscle cells (VSMCs) from the tunica intima into the vessel lumen and subsequent hyperplasia, are key events in the development of atherosclerosis. Molecules that can decrease VSMC proliferation have been examined for possible treatments to slow disease advancement. The discovery that heparin suppresses VSMC growth was reported more than 30 years ago (Clowes and Karnovsky, 1977); yet the mechanism by which heparin treatment of VSMCs inhibits their proliferation remains unclear. Heparin blocks PKC-dependent c-fos induction and activation of ERK, a MAPK activated in response to numerous treatments of sub-cultured VSMCs (Castellot et al., 1989;Ottlinger et al., 1993). In addition, heparin treatment results in decreases in cyclin dependent kinase 2 activity by increasing levels of p27kip1(Fasciano et al., 2005). However, sequestration of growth factors is not likely to explain all of the effects of heparin on VSMCs (Blaukovitch et al., 2010;Pukac et al., 1997;Reilly et al., 1989;Savage et al., 2001). VSMCs specifically bind and endocytose heparin (Castellot et al., 1985). LR-90 This specific binding activity, in combination with heparins effects on cell signaling pathways, supports a model whereby heparin binds to cell surface proteins and initiates its own signaling pathways. To identify putative heparin receptor proteins,Patton et al. (1995)produced monoclonal antibodies that specifically inhibit heparin binding to cellsin vitro. These antibodies identified a single heparin-binding cell surface protein and act as agonists of heparin, mimicking both heparins effects on cell signaling and its anti-proliferative effects in cultured VSMCs (Blaukovitch et al., 2010;Savage et al., 2001). MAPK activity is regulated by the LR-90 reversible phosphorylation of specific tyrosine and threonine residues, and active ERK accumulation in the nucleus is critical in cell cycle progression through G1(Brunet et al., 1999) where the sustained ERK activity results in ELK-1 phosphorylation (Shin et al., 2003). Dual-specificity protein phosphatases play important roles in ERK inactivation, and MAPK Phosphatase-1 (MKP-1) localizes to the nucleus (Rohan et al., 1993) where it is able to regulate ERK signaling in VSMCs. Loss of active ERK in the nucleus results in decreased Elk-1 activity (Shin et al., 2003). Our laboratory has demonstrated that heparin and the anti-heparin receptor antibodies increase MKP-1 levels in VSMCs, playing an important role FANCD1 in heparin-induced ERK activity decreases (Blaukovitch et al., 2010). However, the signaling intermediates between heparins interaction with its receptor and induction of MKP-1 expression remain unknown. One possibility is suggested by studies of insulin signaling (Begum et al., 1998;Jacob et al., 2002). These studies report the expression of MKP-1 in VSMCs in response to insulin and insulin-related growth factor (IGF) where insulin and IGF induce the expression of inducible NOS (iNOS), eventually increasing the levels of cGMP in response to the NO activation of soluble guanylyl cyclase. The increase in cGMP levels was shown to be sufficient to induce MKP-1 expression and attenuate ERK activity. Similarly, leptin treatment induces decreased VSMC proliferation, and this depends on iNOS induction (Rodrguez et al., LR-90 2010). As well as in heparin treated cells, p27kipis induced in response to cGMP rises and cGMP-dependent protein kinase (PKG) activity in VSMCs (Sato et al., 2000). Another agent that elevates cGMP in VSMCs is atrial natriuretic factor or peptide (ANF or ANP). Upon ligand binding, the ANP receptor undergoes a conformational change that activates an intracellular guanylate cyclase domain thereby elevating levels of cGMP. Although better known for their role in vasorelaxation, both ANP and cGMP have been shown to decrease the proliferation of VSMCs (Baldini et al., 2002;Tantini et al., 2005). These effects on VSMCs imply that cGMP mediates a coordinated control of acute cellular function. Elevations in cGMP (by cGMP analogues, activators of guanylate cyclase, or ANP) induce MKP-1 expression in smooth muscle, mesangial and endothelial cells by way of PKG (Baldini et al., 2002;Furst et al., 2005;Jacob et al., 2002;Sugimoto et al., 1996). This increase in MKP-1 expression results in a decrease of ERK activation, and thus provides a mechanism for the anti-proliferative activity of ANP in VSMC and mesangial cells (Baldini et al., 2002;Sugimoto et al., 1996;Tantini et al., 2005). As with heparin, ANP treatment induces increases in p27kip1levels (Tete et al., 2001). Heparin and cGMP affect VSMCs similarly. First, both inhibit growth of VSMCs.