Publications by authors named "Susanne Feil"

67 Publications

Novel soluble guanylyl cyclase activators increase glomerular cGMP, induce vasodilation and improve blood flow in the murine kidney.

Br J Pharmacol 2021 Jun 7. Epub 2021 Jun 7.

Bayer AG, Cardiovascular Research, Pharma Research Center, Wuppertal, Germany.

Background And Purpose: Generation of cGMP via NO-sensitive soluble guanylyl cyclase (sGC) has been implicated in the regulation of renal functions. Chronic kidney disease (CKD) is associated with decreased NO bioavailability, increased oxidative stress and oxidation of sGC to its haem-free form, apo-sGC. Apo-sGC cannot be activated by NO, resulting in impaired cGMP signalling that is associated with chronic kidney disease progression. We hypothesised that sGC activators, which activate apo-sGC independently of NO, increase renal cGMP production under conditions of oxidative stress, thereby improving renal blood flow (RBF) and kidney function.

Experimental Approach: Two novel sGC activators, runcaciguat and BAY-543, were tested on murine kidney. We measured cGMP levels in real time in kidney slices of cGMP sensor mice, vasodilation of pre-constricted glomerular arterioles and RBF in isolated perfused kidneys. Experiments were performed at baseline conditions, under L-NAME-induced NO deficiency, and in the presence of oxidative stress induced by ODQ.

Key Results: Mouse glomeruli showed NO-induced cGMP increases. Under baseline conditions, sGC activator did not alter glomerular cGMP concentration or NO-induced cGMP generation. In the presence of ODQ, NO-induced glomerular cGMP signals were markedly reduced, whereas sGC activator induced strong cGMP increases. L-NAME and ODQ pretreated isolated glomerular arterioles were strongly dilated by sGC activator. sGC activator also increased cGMP and RBF in ODQ-perfused kidneys.

Conclusion And Implication: sGC activators increase glomerular cGMP, dilate glomerular arterioles and improve RBF under disease-relevant oxidative stress conditions. Therefore, sGC activators represent a promising class of drugs for chronic kidney disease treatment.
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http://dx.doi.org/10.1111/bph.15586DOI Listing
June 2021

Visualising and understanding cGMP signals in the cardiovascular system.

Br J Pharmacol 2021 Apr 20. Epub 2021 Apr 20.

Interfakultäres Institut für Biochemie, University of Tübingen, Tübingen, Germany.

cGMP is an important signalling molecule in humans. Fluorescent cGMP biosensors have emerged as powerful tools for the sensitive analysis of cGMP pathways at the single-cell level. Here, we briefly outline cGMP's multifaceted role in (patho)physiology and pharmacotherapy. Then we summarise what new insights cGMP imaging has provided into endogenous cGMP signalling and drug action, with a focus on the cardiovascular system. Indeed, the use of cGMP biosensors has led to several conceptual advances, such as the discovery of local, intercellular and mechanosensitive cGMP signals. Importantly, single-cell imaging can provide valuable information about the heterogeneity of cGMP signals within and between individual cells of an isolated cell population or tissue. We also discuss current challenges and future directions of cGMP imaging, such as the direct visualisation of cGMP microdomains, simultaneous monitoring of cGMP and other signalling molecules and, ultimately, cGMP imaging in tissues and animals under close-to-native conditions.
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http://dx.doi.org/10.1111/bph.15500DOI Listing
April 2021

Olinciguat, a stimulator of soluble guanylyl cyclase, attenuates inflammation, vaso-occlusion and nephropathy in mouse models of sickle cell disease.

Br J Pharmacol 2021 09 30;178(17):3463-3475. Epub 2021 May 30.

Cyclerion Therapeutics Inc., Boston, Massachusetts, USA.

Background And Purpose: Reduced bioavailability of NO, a hallmark of sickle cell disease (SCD), contributes to intravascular inflammation, vasoconstriction, vaso-occlusion and organ damage observed in SCD patients. Soluble guanylyl cyclase (sGC) catalyses synthesis of cGMP in response to NO. cGMP-amplifying agents, including NO donors and phosphodiesterase 9 inhibitors, alleviate TNFα-induced inflammation in wild-type C57BL/6 mice and in 'humanised' mouse models of SCD.

Experimental Approach: Effects of the sGC stimulator olinciguat on intravascular inflammation and renal injury were studied in acute (C57BL6 and Berkeley mice) and chronic (Townes mice) mouse models of TNFα-induced and systemic inflammation associated with SCD.

Key Results: Acute treatment with olinciguat attenuated increases in plasma biomarkers of endothelial cell activation and leukocyte-endothelial cell interactions in TNFα-challenged mice. Co-treatment with hydroxyurea, an FDA-approved SCD therapeutic agent, further augmented the anti-inflammatory effect of olinciguat. In the Berkeley mouse model of TNFα-induced vaso-occlusive crisis, a single dose of olinciguat attenuated leukocyte-endothelial cell interactions, improved blood flow and prolonged survival time compared to vehicle-treated mice. In Townes SCD mice, plasma biomarkers of inflammation and endothelial cell activation were lower in olinciguat- than in vehicle-treated mice. In addition, kidney mass, water consumption, 24-h urine excretion, plasma levels of cystatin C and urinary excretion of N-acetyl-β-d-glucosaminidase and neutrophil gelatinase-associated lipocalin were lower in Townes mice treated with olinciguat than in vehicle-treated mice.

Conclusion And Implications: Our results suggest that the sGC stimulator olinciguat attenuates inflammation, vaso-occlusion and kidney injury in mouse models of SCD and systemic inflammation.
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http://dx.doi.org/10.1111/bph.15492DOI Listing
September 2021

Publisher Correction: A shear-dependent NO-cGMP-cGKI cascade in platelets acts as an auto-regulatory brake of thrombosis.

Nat Commun 2018 11 20;9(1):4969. Epub 2018 Nov 20.

Interfakultäres Institut für Biochemie, University of Tübingen, 72076, Tübingen, Germany.

The original version of this Article contained an error in the description of Supplementary Movie 4, in which the final sentence was inadvertently truncated. The HTML has been updated to include a corrected version of the 'Description of Additional Supplementary Files' file.
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http://dx.doi.org/10.1038/s41467-018-07409-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6244154PMC
November 2018

A shear-dependent NO-cGMP-cGKI cascade in platelets acts as an auto-regulatory brake of thrombosis.

Nat Commun 2018 10 16;9(1):4301. Epub 2018 Oct 16.

Interfakultäres Institut für Biochemie, University of Tübingen, 72076, Tübingen, Germany.

Mechanisms that limit thrombosis are poorly defined. One of the few known endogenous platelet inhibitors is nitric oxide (NO). NO activates NO sensitive guanylyl cyclase (NO-GC) in platelets, resulting in an increase of cyclic guanosine monophosphate (cGMP). Here we show, using cGMP sensor mice to study spatiotemporal dynamics of platelet cGMP, that NO-induced cGMP production in pre-activated platelets is strongly shear-dependent. We delineate a new mode of platelet-inhibitory mechanotransduction via shear-activated NO-GC followed by cGMP synthesis, activation of cGMP-dependent protein kinase I (cGKI), and suppression of Ca signaling. Correlative profiling of cGMP dynamics and thrombus formation in vivo indicates that high cGMP concentrations in shear-exposed platelets at the thrombus periphery limit thrombosis, primarily through facilitation of thrombus dissolution. We propose that an increase in shear stress during thrombus growth activates the NO-cGMP-cGKI pathway, which acts as an auto-regulatory brake to prevent vessel occlusion, while preserving wound closure under low shear.
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http://dx.doi.org/10.1038/s41467-018-06638-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6191445PMC
October 2018

cGMP-dependent protein kinase I (cGKI) modulates human hepatic stellate cell activation.

Metabolism 2018 11 6;88:22-30. Epub 2018 Sep 6.

Department of Internal Medicine IV, Division of Endocrinology, Diabetology, Angiology, Nephrology and Clinical Chemistry, University Hospital Tübingen, Otfried-Müller-Str 10, 72076 Tübingen, Germany; Institute for Diabetes Research and Metabolic Diseases, Helmholtz Center Munich, University of Tübingen, Otfried-Müller-Str 10, 72076 Tübingen, Germany; German Center for Diabetes Research (DZD e.V.), Ingolstädter Landstraße 1, 85764 Neuherberg, Germany.

Background: The activation of hepatic stellate cells (HSCs) plays a crucial role in liver fibrosis, however the role of HSCs is less understood in hepatic insulin resistance. Since in the liver cGMP-dependent protein kinase I (cGKI) was detected in HSC but not in hepatocytes, and cGKI-deficient mice that express cGKI selectively in smooth muscle but not in other cell types (cGKI-SM mice) displayed hepatic insulin resistance, we hypothesized that cGKI modulates HSC activation and insulin sensitivity.

Materials And Methods: To study stellate cell activation in cGKI-SM mice, retinol storage and gene expression were studied. Moreover, in the human stellate cell line LX2, the consequences of cGKI-silencing on gene expression were investigated. Finally, cGKI expression was examined in human liver biopsies covering a wide range of liver fat content.

Results: Retinyl-ester concentrations in the liver of cGKI-SM mice were lower compared to wild-type animals, which was associated with disturbed expression of genes involved in retinol metabolism and inflammation. cGKI-silenced LX2 cells showed an mRNA expression profile of stellate cell activation, altered matrix degradation and activated chemokine expression. On the other hand, activation of LX2 cells suppressed cGKI expression. In accordance with this finding, in human liver biopsies, we observed a negative correlation between cGKI mRNA and liver fat content.

Conclusions: These results suggest that the lack of cGKI possibly leads to stellate cell activation, which stimulates chemokine expression and activates inflammatory processes, which could disturb hepatic insulin sensitivity.
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http://dx.doi.org/10.1016/j.metabol.2018.09.001DOI Listing
November 2018

cGMP Signaling and Vascular Smooth Muscle Cell Plasticity.

J Cardiovasc Dev Dis 2018 04 19;5(2). Epub 2018 Apr 19.

Interfaculty Institute of Biochemistry, University of Tübingen, 72076 Tübingen, Germany.

Cyclic GMP regulates multiple cell types and functions of the cardiovascular system. This review summarizes the effects of cGMP on the growth and survival of vascular smooth muscle cells (VSMCs), which display remarkable phenotypic plasticity during the development of vascular diseases, such as atherosclerosis. Recent studies have shown that VSMCs contribute to the development of atherosclerotic plaques by clonal expansion and transdifferentiation to macrophage-like cells. VSMCs express a variety of cGMP generators and effectors, including NO-sensitive guanylyl cyclase (NO-GC) and cGMP-dependent protein kinase type I (cGKI), respectively. According to the traditional view, cGMP inhibits VSMC proliferation, but this concept has been challenged by recent findings supporting a stimulatory effect of the NO-cGMP-cGKI axis on VSMC growth. Here, we summarize the relevant studies with a focus on VSMC growth regulation by the NO-cGMP-cGKI pathway in cultured VSMCs and mouse models of atherosclerosis, restenosis, and angiogenesis. We discuss potential reasons for inconsistent results, such as the use of genetic versus pharmacological approaches and primary versus subcultured cells. We also explore how modern methods for cGMP imaging and cell tracking could help to improve our understanding of cGMP’s role in vascular plasticity. We present a revised model proposing that cGMP promotes phenotypic switching of contractile VSMCs to VSMC-derived plaque cells in atherosclerotic lesions. Regulation of vascular remodeling by cGMP is not only an interesting new therapeutic strategy, but could also result in side effects of clinically used cGMP-elevating drugs.
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http://dx.doi.org/10.3390/jcdd5020020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023364PMC
April 2018

The Absence of Sensory Axon Bifurcation Affects Nociception and Termination Fields of Afferents in the Spinal Cord.

Front Mol Neurosci 2018 8;11:19. Epub 2018 Feb 8.

Developmental Neurobiology, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany.

A cGMP signaling cascade composed of C-type natriuretic peptide, the guanylyl cyclase receptor Npr2 and cGMP-dependent protein kinase I (cGKI) controls the bifurcation of sensory axons upon entering the spinal cord during embryonic development. However, the impact of axon bifurcation on sensory processing in adulthood remains poorly understood. To investigate the functional consequences of impaired axon bifurcation during adult stages we generated conditional mouse mutants of Npr2 and cGKI ( and ) that lack sensory axon bifurcation in the absence of additional phenotypes observed in the global knockout mice. Cholera toxin labeling in digits of the hind paw demonstrated an altered shape of sensory neuron termination fields in the spinal cord of conditional Npr2 mouse mutants. Behavioral testing of both sexes indicated that noxious heat sensation and nociception induced by chemical irritants are impaired in the mutants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are not affected. Recordings from C-fiber nociceptors in the hind limb skin showed that Npr2 function was not required to maintain normal heat sensitivity of peripheral nociceptors. Thus, the altered behavioral responses to noxious heat found in mice is not due to an impaired C-fiber function. Overall, these data point to a critical role of axonal bifurcation for the processing of pain induced by heat or chemical stimuli.
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http://dx.doi.org/10.3389/fnmol.2018.00019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5809486PMC
February 2018

Cre/lox-assisted non-invasive in vivo tracking of specific cell populations by positron emission tomography.

Nat Commun 2017 09 5;8(1):444. Epub 2017 Sep 5.

Interfakultäres Institut für Biochemie, University of Tübingen, 72076 Tübingen, Germany.

Many pathophysiological processes are associated with proliferation, migration or death of distinct cell populations. Monitoring specific cell types and their progeny in a non-invasive, longitudinal and quantitative manner is still challenging. Here we show a novel cell-tracking system that combines Cre/lox-assisted cell fate mapping with a thymidine kinase (sr39tk) reporter gene for cell detection by positron emission tomography (PET). We generate Rosa26-mT/sr39tk PET reporter mice and induce sr39tk expression in platelets, T lymphocytes or cardiomyocytes. As proof of concept, we demonstrate that our mouse model permits longitudinal PET imaging and quantification of T-cell homing during inflammation and cardiomyocyte viability after myocardial infarction. Moreover, Rosa26-mT/sr39tk mice are useful for whole-body characterization of transgenic Cre mice and to detect previously unknown Cre activity. We anticipate that the Cre-switchable PET reporter mice will be broadly applicable for non-invasive long-term tracking of selected cell populations in vivo.Non-invasive cell tracking is a powerful method to visualize cells in vivo under physiological and pathophysiological conditions. Here Thunemann et al. generate a mouse model for in vivo tracking and quantification of specific cell types by combining a PET reporter gene with Cre-dependent activation that can be exploited for any cell population for which a Cre mouse line is available.
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http://dx.doi.org/10.1038/s41467-017-00482-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585248PMC
September 2017

Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4-negative mouse hearts.

FASEB J 2017 04 30;31(4):1620-1638. Epub 2017 Jan 30.

Department of Pharmacology, Toxicology, and Clinical Pharmacy, Institute of Pharmacy, University of Tübingen, Tübingen, Germany;

LIM domain proteins have been identified as essential modulators of cardiac biology and pathology; however, it is unclear which role the cysteine-rich LIM-only protein (CRP)4 plays in these processes. In studying CRP4 mutant mice, we found that their hearts developed normally, but lack of CRP4 exaggerated multiple parameters of the cardiac stress response to the neurohormone angiotensin II (Ang II). Aiming to dissect the molecular details, we found a link between CRP4 and the cardioprotective cGMP pathway, as well as a multiprotein complex comprising well-known hypertrophy-associated factors. ignificant enrichment of the cysteine-rich intestinal protein (CRIP)1 in murine hearts lacking CRP4, as well as severe cardiac defects and premature death of CRIP1 and CRP4 morphant zebrafish embryos, further support the notion that depleting CRP4 is incompatible with a proper cardiac development and function. Together, amplified Ang II signaling identified CRP4 as a novel antiremodeling factor regulated, at least to some extent, by cardiac cGMP.-Straubinger, J., Boldt, K., Kuret, A., Deng, L., Krattenmacher, D., Bork, N., Desch, M., Feil, R., Feil, S., Nemer, M., Ueffing, M., Ruth, P., Just, S., Lukowski, R. Amplified pathogenic actions of angiotensin II in cysteine-rich LIM-only protein 4 negative mouse hearts.
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http://dx.doi.org/10.1096/fj.201601186DOI Listing
April 2017

Structural Basis for Receptor Recognition by the Human CD59-Responsive Cholesterol-Dependent Cytolysins.

Structure 2016 09 4;24(9):1488-98. Epub 2016 Aug 4.

ACRF Rational Drug Discovery Centre, St. Vincent's Institute of Medical Research, Fitzroy, VIC 3065, Australia; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, VIC 3010, Australia. Electronic address:

Cholesterol-dependent cytolysins (CDCs) are a family of pore-forming toxins that punch holes in the outer membrane of eukaryotic cells. Cholesterol serves as the receptor, but a subclass of CDCs first binds to human CD59. Here we describe the crystal structures of vaginolysin and intermedilysin complexed to CD59. These studies, together with small-angle X-ray scattering, reveal that CD59 binds to each at different, though overlapping, sites, consistent with molecular dynamics simulations and binding studies. The CDC consensus undecapeptide motif, which for the CD59-responsive CDCs has a proline instead of a tryptophan in the motif, adopts a strikingly different conformation between the structures; our data suggest that the proline acts as a selectivity switch to ensure CD59-dependent CDCs bind their protein receptor first in preference to cholesterol. The structural data suggest a detailed model of how these water-soluble toxins assemble as prepores on the cell surface.
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http://dx.doi.org/10.1016/j.str.2016.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320943PMC
September 2016

Sildenafil Potentiates a cGMP-Dependent Pathway to Promote Melanoma Growth.

Cell Rep 2016 Mar 10;14(11):2599-610. Epub 2016 Mar 10.

Interfakultäres Institut für Biochemie, University of Tübingen, 72076 Tübingen, Germany. Electronic address:

Sildenafil, an inhibitor of the cGMP-degrading phosphodiesterase 5 that is used to treat erectile dysfunction, has been linked to an increased risk of melanoma. Here, we have examined the potential connection between cGMP-dependent signaling cascades and melanoma growth. Using a combination of biochemical assays and real-time monitoring of melanoma cells, we report a cGMP-dependent growth-promoting pathway in murine and human melanoma cells. We document that C-type natriuretic peptide (CNP), a ligand of the membrane-bound guanylate cyclase B, enhances the activity of cGMP-dependent protein kinase I (cGKI) in melanoma cells by increasing the intracellular levels of cGMP. Activation of this cGMP pathway promotes melanoma cell growth and migration in a p44/42 MAPK-dependent manner. Sildenafil treatment further increases intracellular cGMP concentrations, potentiating activation of this pathway. Collectively, our data identify this cGMP-cGKI pathway as the link between sildenafil usage and increased melanoma risk.
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http://dx.doi.org/10.1016/j.celrep.2016.02.028DOI Listing
March 2016

Platelet-derived HMGB1 is a critical mediator of thrombosis.

J Clin Invest 2015 Dec 9;125(12):4638-54. Epub 2015 Nov 9.

Thrombosis and inflammation are intricately linked in several major clinical disorders, including disseminated intravascular coagulation and acute ischemic events. The damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1) is upregulated by activated platelets in multiple inflammatory diseases; however, the contribution of platelet-derived HMGB1 in thrombosis remains unexplored. Here, we generated transgenic mice with platelet-specific ablation of HMGB1 and determined that platelet-derived HMGB1 is a critical mediator of thrombosis. Mice lacking HMGB1 in platelets exhibited increased bleeding times as well as reduced thrombus formation, platelet aggregation, inflammation, and organ damage during experimental trauma/hemorrhagic shock. Platelets were the major source of HMGB1 within thrombi. In trauma patients, HMGB1 expression on the surface of circulating platelets was markedly upregulated. Moreover, evaluation of isolated platelets revealed that HMGB1 is critical for regulating platelet activation, granule secretion, adhesion, and spreading. These effects were mediated via TLR4- and MyD88-dependent recruitment of platelet guanylyl cyclase (GC) toward the plasma membrane, followed by MyD88/GC complex formation and activation of the cGMP-dependent protein kinase I (cGKI). Thus, we establish platelet-derived HMGB1 as an important mediator of thrombosis and identify a HMGB1-driven link between MyD88 and GC/cGKI in platelets. Additionally, these findings suggest a potential therapeutic target for patients sustaining trauma and other inflammatory disorders associated with abnormal coagulation.
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http://dx.doi.org/10.1172/JCI81660DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4665785PMC
December 2015

Crystal structure of Streptococcus pneumoniae pneumolysin provides key insights into early steps of pore formation.

Sci Rep 2015 Sep 25;5:14352. Epub 2015 Sep 25.

ACRF Rational Drug Discovery Centre, St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia.

Pore-forming proteins are weapons often used by bacterial pathogens to breach the membrane barrier of target cells. Despite their critical role in infection important structural aspects of the mechanism of how these proteins assemble into pores remain unknown. Streptococcus pneumoniae is the world's leading cause of pneumonia, meningitis, bacteremia and otitis media. Pneumolysin (PLY) is a major virulence factor of S. pneumoniae and a target for both small molecule drug development and vaccines. PLY is a member of the cholesterol-dependent cytolysins (CDCs), a family of pore-forming toxins that form gigantic pores in cell membranes. Here we present the structure of PLY determined by X-ray crystallography and, in solution, by small-angle X-ray scattering. The crystal structure reveals PLY assembles as a linear oligomer that provides key structural insights into the poorly understood early monomer-monomer interactions of CDCs at the membrane surface.
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http://dx.doi.org/10.1038/srep14352DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585913PMC
September 2015

Structure of the lysine specific protease Kgp from Porphyromonas gingivalis, a target for improved oral health.

Protein Sci 2015 Jan 3;24(1):162-6. Epub 2014 Dec 3.

ACRF Rational Drug Discovery Centre, St. Vincent's Institute of Medical Research, Fitzroy, Victoria, 3065, Australia.

The oral pathogen Porphyromonas gingivalis is a keystone pathogen in the development of chronic periodontitis. Gingipains, the principle virulence factors of P. gingivalis are multidomain, cell-surface proteins containing a cysteine protease domain. The lysine specific gingipain, Kgp, is a critical virulence factor of P. gingivalis. We have determined the X-ray crystal structure of the lysine-specific protease domain of Kgp to 1.6 Å resolution. The structure provides insights into the mechanism of substrate specificity and catalysis.
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http://dx.doi.org/10.1002/pro.2589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4282422PMC
January 2015

Transdifferentiation of vascular smooth muscle cells to macrophage-like cells during atherogenesis.

Circ Res 2014 Sep 28;115(7):662-7. Epub 2014 Jul 28.

From the Interfakultäres Institut für Biochemie (S.F., F.E., K.S.-O., R.F.), Department of Dermatology (B.F., M.S.), and Pharmakologie, Toxikologie und Klinische Pharmazie (R.L.), University of Tübingen, Tübingen, Germany.

Rationale: Atherosclerosis is a widespread and devastating disease, but the origins of cells within atherosclerotic plaques are not well defined.

Objective: To investigate the specific contribution of vascular smooth muscle cells (SMCs) to atherosclerotic plaque formation by genetic inducible fate mapping in mice.

Methods And Results: Vascular SMCs were genetically pulse-labeled using the tamoxifen-dependent Cre recombinase, CreER(T2), expressed from the endogenous SM22α locus combined with Cre-activatable reporter genes that were integrated into the ROSA26 locus. Mature SMCs in the arterial media were labeled by tamoxifen treatment of young apolipoprotein E-deficient mice before the development of atherosclerosis and then their fate was monitored in older atherosclerotic animals. We found that medial SMCs can undergo clonal expansion and convert to macrophage-like cells that have lost classic SMC marker expression and make up a major component of advanced atherosclerotic lesions.

Conclusions: This study provides strong in vivo evidence for smooth muscle-to-macrophage transdifferentiation and supports an important role of SMC plasticity in atherogenesis. Targeting this type of SMC phenotypic conversion might be a novel strategy for the treatment of atherosclerosis, as well as other diseases with a smooth muscle component.
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http://dx.doi.org/10.1161/CIRCRESAHA.115.304634DOI Listing
September 2014

Genetic inducible fate mapping in adult mice using tamoxifen-dependent Cre recombinases.

Methods Mol Biol 2014 ;1194:113-39

Interfakultäres Institut für Biochemie, Universität Tübingen, Hoppe-Seyler-Str. 4, Tübingen, 72076, Germany.

The Cre/lox site-specific recombination system allows the control of gene activity in space and time in almost any tissue of the mouse. A major technical advance was the development of tamoxifen-dependent Cre recombinases, such as CreER(T2), that can be activated by administration of tamoxifen to the animal. This powerful tool greatly facilitates the study of gene functions and the generation of more realistic animal models of sporadic human diseases. Another important application of tamoxifen-dependent Cre recombinases is genetic inducible fate mapping (GIFM). In GIFM studies, the inducible Cre/lox system is used to genetically label a defined cell population at a selected time by irreversible activation of the expression of a Cre-responsive reporter transgene. Then, marked cells are detected at later time points to determine how the originally labeled progenitors contribute to specific structures and cell types during pre- and postnatal development. GIFM was initially applied during mouse embryogenesis, but is now increasingly used for cell lineage tracing in adult mice under physiological and pathophysiological conditions. Here we describe the design of GIFM experiments in adult mice as exemplified by CreER(T2)-assisted tracing of vascular smooth muscle cells during the development of atherosclerotic lesions. First, we give an overview of reporter transgenes available for genetic cell marking that are expressed from the Rosa26 locus, such as β-galactosidase and fluorescent proteins. Then we present detailed protocols for the generation of experimental mice for GIFM studies, the induction of cell labeling by tamoxifen treatment, and the detection of marked cells in fixed and live tissues. Each section also provides a discussion of limitations and common pitfalls of GIFM experiments. Most of the protocols can be easily adapted to other developmental stages, cell types, Cre recombinases, and reporter transgenes and, thus, can be used as general guidelines for GIFM studies in mice.
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http://dx.doi.org/10.1007/978-1-4939-1215-5_6DOI Listing
March 2015

Alterations in the cerebellar (Phospho)proteome of a cyclic guanosine monophosphate (cGMP)-dependent protein kinase knockout mouse.

Mol Cell Proteomics 2014 Aug 12;13(8):2004-16. Epub 2014 Jun 12.

From ‡Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands; §Netherlands Proteomics Centre, Padualaan 8, 3584 CH Utrecht, The Netherlands;

The cyclic nucleotide cyclic guanosine monophosphate (cGMP) plays an important role in learning and memory, but its signaling mechanisms in the mammalian brain are not fully understood. Using mass-spectrometry-based proteomics, we evaluated how the cerebellum adapts its (phospho)proteome in a knockout mouse model of cGMP-dependent protein kinase type I (cGKI). Our data reveal that a small subset of proteins in the cerebellum (∼3% of the quantified proteins) became substantially differentially expressed in the absence of cGKI. More changes were observed at the phosphoproteome level, with hundreds of sites being differentially phosphorylated between wild-type and knockout cerebellum. Most of these phosphorylated sites do not represent known cGKI substrates. An integrative computational network analysis of the data indicated that the differentially expressed proteins and proteins harboring differentially phosphorylated sites largely belong to a tight network in the Purkinje cells of the cerebellum involving important cGMP/cAMP signaling nodes (e.g. PDE5 and PKARIIβ) and Ca(2+) signaling (e.g. SERCA3). In this way, removal of cGKI could be linked to impaired cerebellar long-term depression at Purkinje cell synapses. In addition, we were able to identify a set of novel putative (phospho)proteins to be considered in this network. Overall, our data improve our understanding of cerebellar cGKI signaling and suggest novel players in cGKI-regulated synaptic plasticity.
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http://dx.doi.org/10.1074/mcp.M113.035154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4125733PMC
August 2014

Structural studies of Streptococcus pyogenes streptolysin O provide insights into the early steps of membrane penetration.

J Mol Biol 2014 Feb 3;426(4):785-92. Epub 2013 Dec 3.

ACRF Rational Drug Discovery Centre, Biota Structural Biology Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria 3010, Australia. Electronic address:

Cholesterol-dependent cytolysins (CDCs) are a large family of bacterial toxins that exhibit a dependence on the presence of membrane cholesterol in forming large pores in cell membranes. Significant changes in the three-dimensional structure of these toxins are necessary to convert the soluble monomeric protein into a membrane pore. We have determined the crystal structure of the archetypical member of the CDC family, streptolysin O (SLO), a virulence factor from Streptococcus pyogenes. The overall fold is similar to previously reported CDC structures, although the C-terminal domain is in a different orientation with respect to the rest of the molecule. Surprisingly, a signature stretch of CDC sequence called the undecapeptide motif, a key region involved in membrane recognition, adopts a very different structure in SLO to that of the well-characterized CDC perfringolysin O (PFO), although the sequences in this region are identical. An analysis reveals that, in PFO, there are complementary interactions between the motif and the rest of domain 4 that are lost in SLO. Molecular dynamics simulations suggest that the loss of a salt bridge in SLO and a cation-pi interaction are determining factors in the extended conformation of the motif, which in turn appears to result in a greater flexibility of the neighboring L1 loop that houses a cholesterol-sensing motif. These differences may explain the differing abilities of SLO and PFO to efficiently penetrate target cell membranes in the first step of toxin insertion into the membrane.
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http://dx.doi.org/10.1016/j.jmb.2013.11.020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4323271PMC
February 2014

Transgenic mice for cGMP imaging.

Circ Res 2013 Aug 25;113(4):365-71. Epub 2013 Jun 25.

Interfakultäres Institut für Biochemie, Universität Tübingen, Tübingen, Germany.

Rationale: Cyclic GMP (cGMP) is an important intracellular signaling molecule in the cardiovascular system, but its spatiotemporal dynamics in vivo is largely unknown.

Objective: To generate and characterize transgenic mice expressing the fluorescence resonance energy transfer-based ratiometric cGMP sensor, cGMP indicator with an EC50 of 500 nmol/L (cGi500), in cardiovascular tissues.

Methods And Results: Mouse lines with smooth muscle-specific or ubiquitous expression of cGi500 were generated by random transgenesis using an SM22α promoter fragment or by targeted integration of a Cre recombinase-activatable expression cassette driven by the cytomegalovirus early enhancer/chicken β-actin/β-globin promoter into the Rosa26 locus, respectively. Primary smooth muscle cells isolated from aorta, bladder, and colon of cGi500 mice showed strong sensor fluorescence. Basal cGMP concentrations were < 100 nmol/L, whereas stimulation with cGMP-elevating agents such as 2-(N,N-diethylamino)-diazenolate-2-oxide diethylammonium salt (DEA/NO) or the natriuretic peptides, atrial natriuretic peptide, and C-type natriuretic peptide evoked fluorescence resonance energy transfer changes corresponding to cGMP peak concentrations of ≈ 3 µmol/L. However, different types of smooth muscle cells had different sensitivities of their cGMP responses to DEA/NO, atrial natriuretic peptide, and C-type natriuretic peptide. Robust nitric oxide-induced cGMP transients with peak concentrations of ≈ 1 to > 3 µmol/L could also be monitored in blood vessels of the isolated retina and in the cremaster microcirculation of anesthetized mice. Moreover, with the use of a dorsal skinfold chamber model and multiphoton fluorescence resonance energy transfer microscopy, nitric oxide-stimulated vascular cGMP signals associated with vasodilation were detected in vivo in an acutely untouched preparation.

Conclusions: These cGi500 transgenic mice permit the visualization of cardiovascular cGMP signals in live cells, tissues, and mice under normal and pathological conditions or during pharmacotherapy with cGMP-elevating drugs.
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http://dx.doi.org/10.1161/CIRCRESAHA.113.301063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896241PMC
August 2013

Manipulating the Lewis antigen specificity of the cholesterol-dependent cytolysin lectinolysin.

Front Immunol 2012 5;3:330. Epub 2012 Nov 5.

Biota Structural Biology Laboratory and Australian Cancer Research Foundation Rational Drug Discovery Centre, St. Vincent's Institute of Medical Research Fitzroy, VIC, Australia.

The cholesterol-dependent cytolysins (CDCs) attack cells by punching large holes in their membranes. Lectinolysin from Streptococcus mitis is unique among CDCs due to the presence of an N-terminal lectin domain that enhances the pore-forming activity of the toxin. We recently determined the crystal structures of the lectin domain in complex with various glycans. These structures revealed the molecular basis for the Lewis antigen specificity of the toxin. Based on this information we have used in silico molecular modeling to design a mutant toxin, which we predicted would increase its specificity for Lewis y, an antigen found on the surface of cancer cells. Surprisingly, we found by surface plasmon resonance binding experiments that the resultant mutant lectin domain exhibited higher specificity for Lewis b antigens instead. We then undertook comparative crystallographic and molecular dynamics simulation studies of the wild-type and mutant lectin domains to understand the molecular basis for the disparity between the theoretical and experimental results. The crystallographic results revealed that the net number of interactions between Lewis y and wild-type versus mutant was unchanged whereas there was a loss of a hydrogen bond between mutant and Lewis b compared to wild-type. In contrast, the molecular dynamics studies revealed that the Lewis b antigen spent more time in the binding pocket of the mutant compared to wild-type and the reverse was true for Lewis y. The results of these simulation studies are consistent with the conclusions drawn from the surface plasmon resonance studies. This work is part of a program to engineer lectinolysin so that it will target and kill specific cells in human diseases.
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http://dx.doi.org/10.3389/fimmu.2012.00330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3500998PMC
November 2012

An Orally Available 3-Ethoxybenzisoxazole Capsid Binder with Clinical Activity against Human Rhinovirus.

ACS Med Chem Lett 2012 Apr 13;3(4):303-7. Epub 2012 Feb 13.

Biota Holdings Ltd. , 10/585 Blackburn Road, Notting Hill, Victoria 3168, Australia ; Biota Structural Biology Laboratory, St. Vincent's Institute , 9 Princes Street, Fitzroy, Victoria 3065, Australia.

Respiratory infections caused by human rhinovirus are responsible for severe exacerbations of underlying clinical conditions such as asthma in addition to their economic cost in terms of lost working days due to illness. While several antiviral compounds for treating rhinoviral infections have been discovered, none have succeeded, to date, in reaching approval for clinical use. We have developed a potent, orally available rhinovirus inhibitor 6 that has progressed through early clinical trials. The compound shows favorable pharmacokinetic and activity profiles and has a confirmed mechanism of action through crystallographic studies of a rhinovirus-compound complex. The compound has now progressed to phase IIb clinical studies of its effect on natural rhinovirus infection in humans.
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http://dx.doi.org/10.1021/ml2002955DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4025830PMC
April 2012

Presynaptically localized cyclic GMP-dependent protein kinase 1 is a key determinant of spinal synaptic potentiation and pain hypersensitivity.

PLoS Biol 2012 13;10(3):e1001283. Epub 2012 Mar 13.

Pharmacology Institute, University of Heidelberg, Heidelberg, Germany.

Synaptic long-term potentiation (LTP) at spinal neurons directly communicating pain-specific inputs from the periphery to the brain has been proposed to serve as a trigger for pain hypersensitivity in pathological states. Previous studies have functionally implicated the NMDA receptor-NO pathway and the downstream second messenger, cGMP, in these processes. Because cGMP can broadly influence diverse ion-channels, kinases, and phosphodiesterases, pre- as well as post-synaptically, the precise identity of cGMP targets mediating spinal LTP, their mechanisms of action, and their locus in the spinal circuitry are still unclear. Here, we found that Protein Kinase G1 (PKG-I) localized presynaptically in nociceptor terminals plays an essential role in the expression of spinal LTP. Using the Cre-lox P system, we generated nociceptor-specific knockout mice lacking PKG-I specifically in presynaptic terminals of nociceptors in the spinal cord, but not in post-synaptic neurons or elsewhere (SNS-PKG-I(-/-) mice). Patch clamp recordings showed that activity-induced LTP at identified synapses between nociceptors and spinal neurons projecting to the periaqueductal grey (PAG) was completely abolished in SNS-PKG-I(-/-) mice, although basal synaptic transmission was not affected. Analyses of synaptic failure rates and paired-pulse ratios indicated a role for presynaptic PKG-I in regulating the probability of neurotransmitter release. Inositol 1,4,5-triphosphate receptor 1 and myosin light chain kinase were recruited as key phosphorylation targets of presynaptic PKG-I in nociceptive neurons. Finally, behavioural analyses in vivo showed marked defects in SNS-PKG-I(-/-) mice in several models of activity-induced nociceptive hypersensitivity, and pharmacological studies identified a clear contribution of PKG-I expressed in spinal terminals of nociceptors. Our results thus indicate that presynaptic mechanisms involving an increase in release probability from nociceptors are operational in the expression of synaptic LTP on spinal-PAG projection neurons and that PKG-I localized in presynaptic nociceptor terminals plays an essential role in this process to regulate pain sensitivity.
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http://dx.doi.org/10.1371/journal.pbio.1001283DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302842PMC
July 2012

Structure of the lectin regulatory domain of the cholesterol-dependent cytolysin lectinolysin reveals the basis for its lewis antigen specificity.

Structure 2012 Feb;20(2):248-58

Biota Structural Biology Laboratory, St. Vincent's Institute of Medical Research, Fitzroy, Victoria 3065, Australia.

The cholesterol-dependent cytolysins (CDCs) punch holes in target cell membranes through a highly regulated process. Streptococcus mitis lectinolysin (LLY) exhibits another layer of regulation with a lectin domain that enhances the pore-forming activity of the toxin. We have determined the crystal structures of the lectin domain by itself and in complex with various glycans that reveal the molecular basis for the Lewis antigen specificity of LLY. A small-angle X-ray scattering study of intact LLY reveals the molecule is flat and elongated with the lectin domain oriented so that the Lewis antigen-binding site is exposed. We suggest that the lectin domain enhances the pore-forming activity of LLY by concentrating toxin molecules at fucose-rich sites on membranes, thus promoting the formation of prepore oligomers on the surface of susceptible cells.
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http://dx.doi.org/10.1016/j.str.2011.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682648PMC
February 2012

cGMP-Prkg1 signaling and Pde5 inhibition shelter cochlear hair cells and hearing function.

Nat Med 2012 Jan 22;18(2):252-9. Epub 2012 Jan 22.

University of Tübingen, Department of Otolaryngology, Tübingen Hearing Research Centre, Molecular Physiology of Hearing, Tübingen, Germany.

Noise-induced hearing loss (NIHL) is a global health hazard with considerable pathophysiological and social consequences that has no effective treatment. In the heart, lung and other organs, cyclic guanosine monophosphate (cGMP) facilitates protective processes in response to traumatic events. We therefore analyzed NIHL in mice with a genetic deletion of the gene encoding cGMP-dependent protein kinase type I (Prkg1) and found a greater vulnerability to and markedly less recovery from NIHL in these mice as compared to mice without the deletion. Prkg1 was expressed in the sensory cells and neurons of the inner ear of wild-type mice, and its expression partly overlapped with the expression profile of cGMP-hydrolyzing phosphodiesterase 5 (Pde5). Treatment of rats and wild-type mice with the Pde5 inhibitor vardenafil almost completely prevented NIHL and caused a Prkg1-dependent upregulation of poly (ADP-ribose) in hair cells and the spiral ganglion, suggesting an endogenous protective cGMP-Prkg1 signaling pathway that culminates in the activation of poly (ADP-ribose) polymerase. These data suggest vardenafil or related drugs as possible candidates for the treatment of NIHL.
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http://dx.doi.org/10.1038/nm.2634DOI Listing
January 2012

Thiophene inhibitors of PDE4: crystal structures show a second binding mode at the catalytic domain of PDE4D2.

Bioorg Med Chem Lett 2011 Dec 5;21(23):7089-93. Epub 2011 Oct 5.

Medicinal Chemistry & Drug Action, Monash Institute of Pharmaceutical Sciences, Monash University (Parkville campus), Parkville, Victoria, Australia.

PDE4 inhibitors have been identified as therapeutic targets for a variety of conditions, particularly inflammatory diseases. We have serendipitously identified a novel class of phosphodiesterase 4 (PDE4) inhibitor during a study to discover antagonists of the parathyroid hormone receptor. X-ray crystallographic studies of PDE4D2 complexed to four potent inhibitors reveal the atomic details of how they inhibit the enzyme and a notable contrast to another recently reported thiophene-based inhibitor.
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http://dx.doi.org/10.1016/j.bmcl.2011.09.109DOI Listing
December 2011

Fumarates improve psoriasis and multiple sclerosis by inducing type II dendritic cells.

J Exp Med 2011 Oct 10;208(11):2291-303. Epub 2011 Oct 10.

Department of Dermatology, University Medical Center and 2 Interfaculty Institute for Biochemistry, Eberhard Karls University Tübingen, Tübingen, Germany.

Fumarates improve multiple sclerosis (MS) and psoriasis, two diseases in which both IL-12 and IL-23 promote pathogenic T helper (Th) cell differentiation. However, both diseases show opposing responses to most established therapies. First, we show in humans that fumarate treatment induces IL-4-producing Th2 cells in vivo and generates type II dendritic cells (DCs) that produce IL-10 instead of IL-12 and IL-23. In mice, fumarates also generate type II DCs that induce IL-4-producing Th2 cells in vitro and in vivo and protect mice from experimental autoimmune encephalomyelitis. Type II DCs result from fumarate-induced glutathione (GSH) depletion, followed by increased hemoxygenase-1 (HO-1) expression and impaired STAT1 phosphorylation. Induced HO-1 is cleaved, whereupon the N-terminal fragment of HO-1 translocates into the nucleus and interacts with AP-1 and NF-κB sites of the IL-23p19 promoter. This interaction prevents IL-23p19 transcription without affecting IL-12p35, whereas STAT1 inactivation prevents IL-12p35 transcription without affecting IL-23p19. As a consequence, GSH depletion by small molecules such as fumarates induces type II DCs in mice and in humans that ameliorate inflammatory autoimmune diseases. This therapeutic approach improves Th1- and Th17-mediated autoimmune diseases such as psoriasis and MS by interfering with IL-12 and IL-23 production.
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http://dx.doi.org/10.1084/jem.20100977DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3201195PMC
October 2011

HCN3 contributes to the ventricular action potential waveform in the murine heart.

Circ Res 2011 Oct 8;109(9):1015-23. Epub 2011 Sep 8.

Center for Integrated Protein Science CIPS-M, Department Pharmazie, Ludwig-Maximilians-Universität München, München, Germany.

Rationale: The hyperpolarization-activated current I(h) that is generated by hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) plays a key role in the control of pacemaker activity in sinoatrial node cells of the heart. By contrast, it is unclear whether I(h) is also relevant for normal function of cardiac ventricles.

Objective: To study the role of the HCN3-mediated component of ventricular I(h) in normal ventricular function.

Methods And Results: To test the hypothesis that HCN3 regulates the ventricular action potential waveform, we have generated and analyzed a HCN3-deficient mouse line. At basal heart rate, mice deficient for HCN3 displayed a profound increase in the T-wave amplitude in telemetric electrocardiographic measurements. Action potential recordings on isolated ventricular myocytes indicate that this effect was caused by an acceleration of the late repolarization phase in epicardial myocytes. Furthermore, the resting membrane potential was shifted to more hyperpolarized potentials in HCN3-deficient mice. Cardiomyocytes of HCN3-deficient mice displayed approximately 30% reduction of total I(h). At physiological ionic conditions, the HCN3-mediated current had a reversal potential of approximately -35 mV and displayed ultraslow deactivation kinetics.

Conclusions: We propose that HCN3 together with other members of the HCN channel family confer a depolarizing background current that regulates ventricular resting potential and counteracts the action of hyperpolarizing potassium currents in late repolarization. In conclusion, our data indicate that HCN3 plays an important role in shaping the cardiac action potential waveform.
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http://dx.doi.org/10.1161/CIRCRESAHA.111.246173DOI Listing
October 2011

Genetic ablation of cGMP-dependent protein kinase type I causes liver inflammation and fasting hyperglycemia.

Diabetes 2011 May 4;60(5):1566-76. Epub 2011 Apr 4.

Department of Internal Medicine, Division of Endocrinology, Diabetology, Vascular Disease, Nephrology and Clinical Chemistry, University of Tübingen, Tübingen, Germany.

Objective: The nitric oxide/cGMP/cGMP-dependent protein kinase type I (cGKI) signaling pathway regulates cell functions that play a pivotal role in the pathogenesis of type 2 diabetes. However, the impact of a dysfunction of this pathway for glucose metabolism in vivo is unknown.

Research Design And Methods: The expression of cGKI in tissues relevant to insulin action was analyzed by immunohistochemistry. The metabolic consequences of a genetic deletion of cGKI were studied in mice that express cGKI selectively in smooth muscle but not in other cell types (cGKI-SM mice).

Results: In wild-type mice, cGKI protein was detected in hepatic stellate cells, but not in hepatocytes, skeletal muscle, fat cells, or pancreatic β-cells. Compared with control animals, cGKI-SM mice had higher energy expenditure in the light phase associated with lower body weight and fat mass and increased insulin sensitivity. Mutant mice also showed higher fasting glucose levels, whereas insulin levels and intraperitoneal glucose tolerance test results were similar to those in control animals. Interleukin (IL)-6 signaling was strongly activated in the liver of cGKI-SM mice as demonstrated by increased levels of IL-6, phospho-signal transducer and activator of transcription 3 (Tyr 705), suppressor of cytokine signaling-3, and serum amyloid A2. Insulin-stimulated tyrosine phosphorylation of the insulin receptor in the liver was impaired in cGKI-SM mice. The fraction of Mac-2-positive macrophages in the liver was significantly higher in cGKI-SM mice than in control mice. In contrast with cGKI-SM mice, conditional knockout mice lacking cGKI only in the nervous system were normal with respect to body weight, energy expenditure, fasting glucose, IL-6, and insulin action in the liver.

Conclusions: Genetic deletion of cGKI in non-neuronal cells results in a complex metabolic phenotype, including liver inflammation and fasting hyperglycemia. Loss of cGKI in hepatic stellate cells may affect liver metabolism via a paracrine mechanism that involves enhanced macrophage infiltration and IL-6 signaling.
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http://dx.doi.org/10.2337/db10-0760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3292332PMC
May 2011

Proteins: membrane binding and pore formation. Introduction.

Adv Exp Med Biol 2010 ;677:1-13

St. Vincent's Institute of Medical Research, Fitzroy, Victoria, Australia.

Pore-forming proteins (PFPs) possess the intriguing property that they can exist either in a stable water-soluble state or as an integral membrane pore. These molecules can undergo large conformational changes in converting between these two states. Much of what we know about how these proteins change their shape comes from work on bacterial toxins and increasingly, in more recent years, on toxins from other organisms. Surprisingly, a number of pore-forming proteins have recently been characterised that appear to have adopted similar stratagies to toxins for binding and inserting into biological membranes.
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August 2010
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