Yvonne Keim

Publications of Yvonne Keim

• Fluorescence dequenching makes haem-free soluble guanylate cyclase detectable in living cells.

  Authors: Linda S Hoffmann, Peter M Schmidt, Yvonne Keim, Carsten Hoffmann, Harald H H W Schmidt, Johannes-Peter Stasch

  PloS one. 01/2011; 6(8):e23596.

  In cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitiveIn cardiovascular disease, the protective NO/sGC/cGMP signalling-pathway is impaired due to a decreased pool of NO-sensitive haem-containing sGC accompanied by a reciprocal increase in NO-insensitive haem-free sGC. However, no direct method to detect cellular haem-free sGC other than its activation by the new therapeutic class of haem mimetics, such as BAY 58-2667, is available. Here we show that fluorescence dequenching, based on the interaction of the optical active prosthetic haem group and the attached biarsenical fluorophor FlAsH can be used to detect changes in cellular sGC haem status. The partly overlap of the emission spectrum of haem and FlAsH allows energy transfer from the fluorophore to the haem which reduces the intensity of FlAsH fluorescence. Loss of the prosthetic group, e.g. by oxidative stress or by replacement with the haem mimetic BAY 58-2667, prevented the energy transfer resulting in increased fluorescence. Haem loss was corroborated by an observed decrease in NO-induced sGC activity, reduced sGC protein levels, and an increased effect of BAY 58-2667. The use of a haem-free sGC mutant and a biarsenical dye that was not quenched by haem as controls further validated that the increase in fluorescence was due to the loss of the prosthetic haem group. The present approach is based on the cellular expression of an engineered sGC variant limiting is applicability to recombinant expression systems. Nevertheless, it allows to monitor sGC's redox regulation in living cells and future enhancements might be able to extend this approach to in vivo conditions.

• Distinct molecular requirements for activation or stabilization of soluble guanylyl cyclase upon haem oxidation-induced degradation

  Authors: Linda Hoffmann, Peter Schmidt, Yvonne Keim, Stefan Schaefer, Harald Schmidt, Johannes-Peter Stasch

  BMC Pharmacology. 01/2009;

• Nitric oxide-independent activation of soluble guanylate cyclase by BAY 60-2770 in experimental liver fibrosis.

  Authors: Andreas Knorr, Claudia Hirth-Dietrich, Cristina Alonso-Alija, Michael Härter, Michael Hahn, Yvonne Keim, Frank Wunder, Johannes-Peter Stasch

  Arzneimittel-Forschung. 02/2008; 58(2):71-80.

  Liver cirrhosis is a chronic disease with high mortality rate and need for effective pharmacological intervention. The fibrotic remodelling of liver tissue is crucially dependent on hepatic stellateLiver cirrhosis is a chronic disease with high mortality rate and need for effective pharmacological intervention. The fibrotic remodelling of liver tissue is crucially dependent on hepatic stellate cell activation. Activation of hepatic stellate cells is reduced by an increase in cyclic guanosine monophosphate (cGMP). Stable cGMP analogues also reduce the contractile response of hepatic stellate cells. However, cGMP production is downregulated in the cirrhotic liver due to the reduced activity of the endothelial nitric oxide synthase. OBJECTIVE: Here we report that the novel activator of soluble guanylate cyclase (sGC), BAY 60-2770 (4-({(4-carboxybutyl) [2- (5-fluoro-2-{[4'-(trifluoromethyl) biphenyl-4-yl]methoxy}phenyl)ethyl] amino}methyl)benzoic acid), which increases the activity of sGC in a nitric oxide-independent manner, attenuates liver fibrosis in two rat models. METHODS: The compound was studied in the pig serum model and the carbon tetrachloride model. Fibrosis was assessed by estimating the increase in fibrous collagen by micromorphometry of histological sections stained with Sirius Red/Fast Green and by measuring total hepatic collagen. RESULTS: BAY 60-2770, on a recombinant sGC reporter cell line, stimulated the luminescence signals with an EC50 value of 5.4 +/- 1.2 nmol/L. In the presence of [1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 10 pmol/L) the EC50 was shifted to 0.39 +/- 0.11 nmol/L. In both fibrosis models, once daily oral administration of BAY 60-2770 concomittantly with the fibrotic stimulus prevented 60-75% of fibrosis, the lowest effective dose being 0.1 mg/kg in the pig serum model and 0.3 mg/kg in the carbon tetrachloride model. The treatment was well tolerated by all animals. The doses used were devoid of any significant influence on systemic blood pressure. Conclusion: Nitric oxide-independent activation of sGC might be an innovative therapeutic approach for the treatment of liver fibrosis of necro-inflammatory and immunological origin.

• Antifibrotic effects of an sGC activator in rat models of liver fibrosis

  Authors: Claudia Hirth-Dietrich, Cristina Alonso-Alija, Michael Härter, Michael Hahn, Yvonne Keim, Frank Wunder, Andreas Knorr, Johannes-Peter Stasch

  BMC Pharmacology. 01/2005;