Abstract-Nitric oxide (NO) is an essential vasodilator. In vascular diseases, oxidative stress attenuates NO signaling by both chemical scavenging of free NO and oxidation and downregulation of its major intracellular receptor, the ␣␤ heterodimeric heme-containing soluble guanylate cyclase (sGC). Oxidation can also induce loss of the heme of sGC, as well as the responsiveness of sGC to NO. sGC activators such as BAY 58-2667 bind to oxidized/heme-free sGC and reactivate the enzyme to exert disease-specific vasodilation. Here, we show that oxidation-induced downregulation of sGC protein extends to isolated blood vessels. Mechanistically, degradation was triggered through sGC ubiquitination and proteasomal degradation. The heme-binding site ligand BAY 58-2667 prevented sGC ubiquitination and stabilized both ␣ and ␤ subunits. Collectively, our data establish oxidation-ubiquitination of sGC as a modulator of NO/cGMP signaling and point to a new mechanism of action for sGC activating vasodilators by stabilizing their receptor, oxidized/heme-free sGC. O ne major risk factor for the development of cardiovascular diseases, such as coronary heart disease, stroke, and myocardial infarction, is an imbalance of the production and elimination of reactive oxygen species, also termed as oxidative stress. [1][2][3] As a consequence, the nitric oxide (NO)/ cGMP signaling cascade is impaired, eg, through the excessive production of superoxide, which reacts with NO in a diffusion-limited reaction, yielding peroxynitrite. 4 The biological impact of NO scavenging is further aggravated by the progressive inhibition and downregulation of the NO receptor soluble guanylate cyclase (sGC). [5][6][7][8][9] Circumstantial evidence has implicated proteasomal pathways in this downregulation of sGC. 10 -12 Conversely, a novel class of sGC activators, represented by BAY 58-2667, are potentiated under oxidative stress conditions and represent thus an entirely new diseasespecific vasodilator class. 13 sGC is a heterodimer consisting of an ␣ and a Fe 2ϩ /hemecontaining ␤ subunit that complexes NO with high affinity and specificity. 14 Binding of NO to the Fe 2ϩ /heme results in allosteric activation of the enzyme and enhanced conversion of GTP into the vasorelaxant and antiproliferative second messenger cGMP. 14,15 In vitro experiments have demonstrated that oxidation of sGC heme to its ferric (Fe 3ϩ ) form by the sGC inhibitor 1H-[1,2,4]-oxadiazolo [3,4-a]quinoxalin-1-one (ODQ) attenuates NO-mediated cGMP production, suggesting that the ferro (Fe 2ϩ ) form of sGC is crucial for activation by NO. 16 -18 In addition, studies with primary endothelial and smooth muscle cells have revealed that, within 24 hours, ODQ causes a dramatic decrease in sGC protein levels. 13 Similar results were obtained with other oxidizing compounds such as methylene blue or the peroxynitrite donor 1,3-morpholino-sydnonimine hydrochloride (SIN-1), indicating that oxidative stress triggers downregulation of sGC protein levels. 13 At present, the molecular mechanisms under...

BackgroundA direct pharmacological stimulation of soluble guanylate cyclase (sGC) is an emerging therapeutic approach to the management of various cardiovascular disorders associated with endothelial dysfunction. Novel sGC stimulators, including riociguat (BAY 63-2521), have a dual mode of action: They sensitize sGC to endogenously produced nitric oxide (NO) and also directly stimulate sGC independently of NO. Little is known about their effects on tissue remodeling and degeneration and survival in experimental malignant hypertension.Methods and ResultsMortality, hemodynamics and biomarkers of tissue remodeling and degeneration were assessed in Dahl salt-sensitive rats maintained on a high salt diet and treated with riociguat (3 or 10 mg/kg/d) for 14 weeks. Riociguat markedly attenuated systemic hypertension, improved systolic heart function and increased survival from 33% to 85%. Histological examination of the heart and kidneys revealed that riociguat significantly ameliorated fibrotic tissue remodeling and degeneration. Correspondingly, mRNA expression of the pro-fibrotic biomarkers osteopontin (OPN), tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and plasminogen activator inhibitor-1 (PAI-1) in the myocardium and the renal cortex was attenuated by riociguat. In addition, riociguat reduced plasma and urinary levels of OPN, TIMP-1, and PAI-1.ConclusionsStimulation of sGC by riociguat markedly improves survival and attenuates systemic hypertension and systolic dysfunction, as well as fibrotic tissue remodeling in the myocardium and the renal cortex in a rodent model of pressure and volume overload. These findings suggest a therapeutic potential of sGC stimulators in diseases associated with impaired cardiovascular and renal functions.

We demonstrate for the first time that the novel sGC stimulator riociguat shows in two independent models of hypertension a potent protection against cardiac and renal target organ damage.