Abnormal vascular smooth muscle (VSM) growth remains an elemental foundation of vasculoproliferative disorders including atherosclerosis and restenosis. Many second messenger, cytokine, and growth factor signals mediate control of VSM growth, and among these is transforming growth factor (TGF)-β1, a pluripotent cytokine with wide-ranging yet often opposite effects in VSM. Cyclic nucleotide signaling also exerts powerful growth control of VSM, and our previous work has helped establish a biological link between cyclic GMP and TGF-β1 in injured carotid arteries. The current study characterized the influence of cyclic GMP on TGF-β1 and its receptor-activated Smad3 in rat primary VSM cells. The heme-dependent soluble guanylate cyclase (sGC) stimulator BAY 41-2272 (BAY41) significantly increased cyclic GMP and site-specific phosphorylation of vasodilator-activated serum phosphoprotein (VASP) in manner indicative of active protein kinase G (PKG) and PKA signaling. Recombinant TGF-β1 (10 ng/ml) significantly stimulated phospho-Smad3 (Ser
423/425
) and decreased inhibitory Smad7 in VSM cell homogenates, and using flow cytometry significantly increased cells in G
2
/M and expression of cyclins D and E and Cdk2 and Cdk4 while decreasing expression of inhibitory p21 and p27 after 24 hours compared to vehicle controls. TGF-β1 also significantly increased cell numbers compared to controls after 48 hours, thus confirming growth promoting capacities of TGF-β1 in VSM. In cell lysates double-sandwich ELISA revealed that BAY41 significantly reduces total and active TGF-β1, and Western analyses showed it significantly decreases total and phospho-Smad3
Ser423/425
expression and reduces MMP-2 and MMP-9 expression and activity (via column zymography) in both cell lysates and conditioned media after 1 and 48 hours. BAY41 also significantly reduced serum- and PDGF-stimulated cell migration between 6 and 18 hours using an in vitro scrape injury and a transwell assay. In comparison, inclusive effects of BAY41 were replicated by its prototype YC-1 and by the heme-independent sGC activator BAY 60-2770. These data clearly support growth protective capacities of cGMP in VSM and propose it operates through attenuation of TGF-β1/Smad3 signaling.
Cyclic GMP-dependent but G-kinase-independent inhibition of Ca2+-dependent Cl−currents by NO donors in cat tracheal smooth muscle
