Publications by authors named "Robert Feil"

206 Publications

Exploring chromatin structural roles of non-coding RNAs at imprinted domains.

Biochem Soc Trans 2021 Aug;49(4):1867-1879

Institute of Molecular Genetics of Montpellier (IGMM), Centre National de Recherche Scientifique (CNRS), Montpellier, France.

Different classes of non-coding RNA (ncRNA) influence the organization of chromatin. Imprinted gene domains constitute a paradigm for exploring functional long ncRNAs (lncRNAs). Almost all express an lncRNA in a parent-of-origin dependent manner. The mono-allelic expression of these lncRNAs represses close by and distant protein-coding genes, through diverse mechanisms. Some control genes on other chromosomes as well. Interestingly, several imprinted chromosomal domains show a developmentally regulated, chromatin-based mechanism of imprinting with apparent similarities to X-chromosome inactivation. At these domains, the mono-allelic lncRNAs show a relatively stable, focal accumulation in cis. This facilitates the recruitment of Polycomb repressive complexes, lysine methyltranferases and other nuclear proteins - in part through direct RNA-protein interactions. Recent chromosome conformation capture and microscopy studies indicate that the focal aggregation of lncRNA and interacting proteins could play an architectural role as well, and correlates with close positioning of target genes. Higher-order chromatin structure is strongly influenced by CTCF/cohesin complexes, whose allelic association patterns and actions may be influenced by lncRNAs as well. Here, we review the gene-repressive roles of imprinted non-coding RNAs, particularly of lncRNAs, and discuss emerging links with chromatin architecture.
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http://dx.doi.org/10.1042/BST20210758DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8421051PMC
August 2021

Zfp57 inactivation illustrates the role of ICR methylation in imprinted gene expression during neural differentiation of mouse ESCs.

Sci Rep 2021 Jul 5;11(1):13802. Epub 2021 Jul 5.

Department of Environmental Biological and Pharmaceutical Sciences and Technologies (DiSTABiF), Università degli Studi della Campania "Luigi Vanvitelli", 81100, Caserta, Italy.

ZFP57 is required to maintain the germline-marked differential methylation at imprinting control regions (ICRs) in mouse embryonic stem cells (ESCs). Although DNA methylation has a key role in genomic imprinting, several imprinted genes are controlled by different mechanisms, and a comprehensive study of the relationship between DMR methylation and imprinted gene expression is lacking. To address the latter issue, we differentiated wild-type and Zfp57 hybrid mouse ESCs into neural precursor cells (NPCs) and evaluated allelic expression of imprinted genes. In mutant NPCs, we observed a reduction of allelic bias of all the 32 genes that were imprinted in wild-type cells, demonstrating that ZFP57-dependent methylation is required for maintaining or acquiring imprinted gene expression during differentiation. Analysis of expression levels showed that imprinted genes expressed from the non-methylated chromosome were generally up-regulated, and those expressed from the methylated chromosome were down-regulated in mutant cells. However, expression levels of several imprinted genes acquiring biallelic expression were not affected, suggesting the existence of compensatory mechanisms that control their RNA level. Since neural differentiation was partially impaired in Zfp57-mutant cells, this study also indicates that imprinted genes and/or non-imprinted ZFP57-target genes are required for proper neurogenesis in cultured ESCs.
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http://dx.doi.org/10.1038/s41598-021-93297-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8257706PMC
July 2021

The C5a/C5a receptor 1 axis controls tissue neovascularization through CXCL4 release from platelets.

Nat Commun 2021 06 7;12(1):3352. Epub 2021 Jun 7.

Cardioimmunology Group, Medical Clinic II, University Heart Center Lübeck, Lübeck, Germany.

Platelets contribute to the regulation of tissue neovascularization, although the specific factors underlying this function are unknown. Here, we identified the complement anaphylatoxin C5a-mediated activation of C5a receptor 1 (C5aR1) on platelets as a negative regulatory mechanism of vessel formation. We showed that platelets expressing C5aR1 exert an inhibitory effect on endothelial cell functions such as migration and 2D and 3D tube formation. Growth factor- and hypoxia-driven vascularization was markedly increased in C5ar1 mice. Platelet-specific deletion of C5aR1 resulted in a proangiogenic phenotype with increased collateralization, capillarization and improved pericyte coverage. Mechanistically, we found that C5a induced preferential release of CXC chemokine ligand 4 (CXCL4, PF4) from platelets as an important antiangiogenic paracrine effector molecule. Interfering with the C5aR1-CXCL4 axis reversed the antiangiogenic effect of platelets both in vitro and in vivo.In conclusion, we identified a mechanism for the control of tissue neovascularization through C5a/C5aR1 axis activation in platelets and subsequent induction of the antiangiogenic factor CXCL4.
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http://dx.doi.org/10.1038/s41467-021-23499-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8185003PMC
June 2021

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 Sep 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

Emerging chromatin structural roles of the methyl-CpG binding protein MeCP2.

Epigenomics 2021 Mar 9;13(6):405-409. Epub 2021 Mar 9.

Institute of Molecular Genetics of Montpellier (IGMM), Centre National de Recherche Scientifique (CNRS), 1919 route de Mende, 34293 Montpellier, France.

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http://dx.doi.org/10.2217/epi-2021-0053DOI Listing
March 2021

Amelanotic B16-F10 Melanoma Compatible with Advanced Three-Dimensional Imaging Modalities.

J Invest Dermatol 2021 Aug 4;141(8):2090-2094.e6. Epub 2021 Mar 4.

Immune Imaging Program, Centenary Institute, Faculty of Medicine and Health, The University of Sydney, Camperdown, Australia; Sydney Medical School, The University of Sydney, Sydney, Australia; Department of Dermatology, Medical University of Vienna, Vienna, Austria.

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http://dx.doi.org/10.1016/j.jid.2021.01.025DOI Listing
August 2021

Analysis of Gene Expression Using lacZ Reporter Mouse Lines.

Methods Mol Biol 2021 ;2224:29-45

Interfaculty Institute of Biochemistry, Eberhard Karls University Tübingen, Tübingen, Germany.

Reporter mice transgenically expressing the bacterial (E. coli) lacZ gene encoding β-galactosidase (β-gal, EC 3.2.1.23) are a versatile and extensively used tool to study gene expression and cell lineage patterns. Enzymatic activity of the β-gal reporter can be effectively visualized at cellular resolution either histochemically using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) or by immunofluorescent detection using a β-gal-specific antibody. Here, we summarize protocols for the localization of β-gal expressing cells in whole embryos or organs as well as in histological tissue sections of lacZ reporter mice and discuss their limitations and common pitfalls.
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http://dx.doi.org/10.1007/978-1-0716-1008-4_2DOI Listing
April 2021

A Role for COMPASS in Germline Chromatin Organization.

Cells 2020 09 8;9(9). Epub 2020 Sep 8.

Laboratory of Biology and Modeling of the Cell (LBMC), CNRS, Ecole Normale Supérieure de Lyon, Université de Lyon, 69007 Lyon, France.

Deposition of histone H3 lysine 4 (H3K4) methylation at promoters is catalyzed by the SET1/COMPASS complex and is associated with context-dependent effects on gene expression and local changes in chromatin organization. The role of SET1/COMPASS in shaping chromosome architecture has not been investigated. Here we used to address this question through a live imaging approach and genetic analysis. Using quantitative FRET (Förster resonance energy transfer)-based fluorescence lifetime imaging microscopy (FLIM) on germ cells expressing histones eGFP-H2B and mCherry-H2B, we find that SET1/COMPASS influences meiotic chromosome organization, with marked effects on the close proximity between nucleosomes. We further show that inactivation of , encoding the SET1 homologue, or CFP-1, encoding the chromatin targeting subunit of COMPASS, enhances germline chromosome organization defects and sterility of condensin-II depleted animals. loss also aggravates germline defects resulting from conditional inactivation of topoisomerase II, another structural component of chromosomes. Expression profiling of mutant germlines revealed only minor transcriptional changes, suggesting that the observed effects are at least partly independent of transcription. Altogether, our results are consistent with a role for SET1/COMPASS in shaping meiotic chromosomes in , together with the non-histone proteins condensin-II and topoisomerase. Given the high degree of conservation, our findings expand the range of functions attributed to COMPASS and suggest a broader role in genome organization in different species.
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http://dx.doi.org/10.3390/cells9092049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7565041PMC
September 2020

Guanylyl Cyclase A/cGMP Signaling Slows Hidden, Age- and Acoustic Trauma-Induced Hearing Loss.

Front Aging Neurosci 2020 9;12:83. Epub 2020 Apr 9.

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

In the inner ear, cyclic guanosine monophosphate (cGMP) signaling has been described as facilitating otoprotection, which was previously observed through elevated cGMP levels achieved by phosphodiesterase 5 inhibition. However, to date, the upstream guanylyl cyclase (GC) subtype eliciting cGMP production is unknown. Here, we show that mice with a genetic disruption of the gene encoding the cGMP generator GC-A, the receptor for atrial and B-type natriuretic peptides, display a greater vulnerability of hair cells to hidden hearing loss and noise- and age-dependent hearing loss. This vulnerability was associated with GC-A expression in spiral ganglia and outer hair cells (OHCs) but not in inner hair cells (IHCs). GC-A knockout mice exhibited elevated hearing thresholds, most pronounced for the detection of high-frequency tones. Deficits in OHC input-output functions in high-frequency regions were already present in young GC-A-deficient mice, with no signs of an accelerated progression of age-related hearing loss or higher vulnerability to acoustic trauma. OHCs in these frequency regions in young GC-A knockout mice exhibited diminished levels of KCNQ4 expression, which is the dominant K channel in OHCs, and decreased activation of poly (ADP-ribose) polymerase-1, an enzyme involved in DNA repair. Further, GC-A knockout mice had IHC synapse impairments and reduced amplitudes of auditory brainstem responses that progressed with age and with acoustic trauma, in contrast to OHCs, when compared to GC-A wild-type littermates. We conclude that GC-A/cGMP-dependent signaling pathways have otoprotective functions and GC-A gene disruption differentially contributes to hair-cell damage in a healthy, aged, or injured system. Thus, augmentation of natriuretic peptide GC-A signaling likely has potential to overcome hidden and noise-induced hearing loss, as well as presbycusis.
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http://dx.doi.org/10.3389/fnagi.2020.00083DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160671PMC
April 2020

Differential 3D chromatin organization and gene activity in genomic imprinting.

Curr Opin Genet Dev 2020 04 13;61:17-24. Epub 2020 Apr 13.

Institute of Molecular Genetics of Montpellier (IGMM), University of Montpellier, CNRS, Montpellier, France. Electronic address:

Genomic imprinting gives rise to parent-of-origin dependent allelic gene expression. Most imprinted genes cluster in domains where differentially methylated regions (DMRs)-carrying CpG methylation on one parental allele-regulate their activity. Several imprinted DMRs bind CTCF on the non-methylated allele. CTCF structures TADs ('Topologically Associating Domains'), which are structural units of transcriptional regulation. Recent investigations show that imprinted domains are embedded within TADs that are similar on both parental chromosomes. Within these TADs, however, allelic subdomains are structured by combinations of mono-allelic and bi-allelic CTCF binding that guide imprinted expression. This emerging view indicates that imprinted chromosomal domains should be considered at the overarching TAD level, and questions how CTCF integrates with other regulatory proteins and lncRNAs to achieve imprinted transcriptional programs.
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http://dx.doi.org/10.1016/j.gde.2020.03.004DOI Listing
April 2020

Imprinted Small RNAs Unraveled: Maternal MicroRNAs Antagonize a Paternal-Genome-Driven Gene Expression Network.

Mol Cell 2020 04;78(1):3-5

Institute of Molecular Genetics of Montpellier (IGMM), CNRS, University of Montpelllier, 34293 Montpellier, France. Electronic address:

Whipple et al., 2020 describe that, in neurons, a dense cluster of maternally expressed microRNAs post-transcriptionally downregulates several imprinted genes expressed from the paternal genome-an antagonistic mechanism that modulates neuronal functions and provides insights into the evolution of genomic imprinting.
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http://dx.doi.org/10.1016/j.molcel.2020.03.019DOI Listing
April 2020

CTCF modulates allele-specific sub-TAD organization and imprinted gene activity at the mouse Dlk1-Dio3 and Igf2-H19 domains.

Genome Biol 2019 12 12;20(1):272. Epub 2019 Dec 12.

Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, University Paris-sud and University Paris-Saclay, Gif-sur-Yvette, France.

Background: Genomic imprinting is essential for mammalian development and provides a unique paradigm to explore intra-cellular differences in chromatin configuration. So far, the detailed allele-specific chromatin organization of imprinted gene domains has mostly been lacking. Here, we explored the chromatin structure of the two conserved imprinted domains controlled by paternal DNA methylation imprints-the Igf2-H19 and Dlk1-Dio3 domains-and assessed the involvement of the insulator protein CTCF in mouse cells.

Results: Both imprinted domains are located within overarching topologically associating domains (TADs) that are similar on both parental chromosomes. At each domain, a single differentially methylated region is bound by CTCF on the maternal chromosome only, in addition to multiple instances of bi-allelic CTCF binding. Combinations of allelic 4C-seq and DNA-FISH revealed that bi-allelic CTCF binding alone, on the paternal chromosome, correlates with a first level of sub-TAD structure. On the maternal chromosome, additional CTCF binding at the differentially methylated region adds a further layer of sub-TAD organization, which essentially hijacks the existing paternal-specific sub-TAD organization. Perturbation of maternal-specific CTCF binding site at the Dlk1-Dio3 locus, using genome editing, results in perturbed sub-TAD organization and bi-allelic Dlk1 activation during differentiation.

Conclusions: Maternal allele-specific CTCF binding at the imprinted Igf2-H19 and the Dlk1-Dio3 domains adds an additional layer of sub-TAD organization, on top of an existing three-dimensional configuration and prior to imprinted activation of protein-coding genes. We speculate that this allele-specific sub-TAD organization provides an instructive or permissive context for imprinted gene activation during development.
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http://dx.doi.org/10.1186/s13059-019-1896-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6909504PMC
December 2019

Stability and Lability of Parental Methylation Imprints in Development and Disease.

Genes (Basel) 2019 12 2;10(12). Epub 2019 Dec 2.

Institute of Molecular Genetics (IGMM), CNRS, University of Montpellier, 1919 route de Mende, 34293 Montpellier, France.

DNA methylation plays essential roles in mammals. Of particular interest are parental methylation marks that originate from the oocyte or the sperm, and bring about mono-allelic gene expression at defined chromosomal regions. The remarkable somatic stability of these parental imprints in the pre-implantation embryo-where they resist global waves of DNA demethylation-is not fully understood despite the importance of this phenomenon. After implantation, some methylation imprints persist in the placenta only, a tissue in which many genes are imprinted. Again here, the underlying epigenetic mechanisms are not clear. Mouse studies have pinpointed the involvement of transcription factors, covalent histone modifications, and histone variants. These and other features linked to the stability of methylation imprints are instructive as concerns their conservation in humans, in which different congenital disorders are caused by perturbed parental imprints. Here, we discuss DNA and histone methylation imprints, and why unravelling maintenance mechanisms is important for understanding imprinting disorders in humans.
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http://dx.doi.org/10.3390/genes10120999DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6947649PMC
December 2019

Methylation of the C19MC microRNA locus in the placenta: association with maternal and chilhood body size.

Int J Obes (Lond) 2020 01 25;44(1):13-22. Epub 2019 Sep 25.

Department of Pediatrics, Girona Institute for Biomedical Research (IDIBGI), Salt, Spain.

Objectives: To study DNA methylation at the C19MC locus in the placenta and its association with (1) parental body size, (2) transmission of haplotypes for the C19MC rs55765443 SNP, and (3) offspring's body size and/or body composition at birth and in childhood.

Subjects And Methods: Seventy-two pregnant women-infant pairs and 63 fathers were included in the study. Weight and height of mothers, fathers and newborns were registered during pregnancy or at birth (n = 72). Placental DNA methylation at the C19MC imprinting control region (ICR) was quantified by bisulfite pyrosequencing. Genotyping of the SNP was performed using restriction fragment length polymorphisms. The children's body size and composition were reassessed at age 6 years (n = 32).

Results: Lower levels of placental C19MC methylation were associated with increased body size of mother, specifically with higher pregestational and predelivery weights and height of the mother (β from -0.294 to -0.371; R from 0.04 to 0.10 and all p < 0.019), and with higher weight, height, waist and hip circumferences, and fat mass of the child (β from -0.428 to -0.552; R from 0.33 to 0.56 and all p < 0.009). Parental transmission of the SNP did not correlate with an altered placental methylation status at the C19MC ICR.

Conclusions: Increased maternal size is associated with reduced placental C19MC methylation, which, in turn, relate to larger body size of the child.
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http://dx.doi.org/10.1038/s41366-019-0450-9DOI Listing
January 2020

cGMP-dependent protein kinase I in vascular smooth muscle cells improves ischemic stroke outcome in mice.

J Cereb Blood Flow Metab 2019 12 18;39(12):2379-2391. Epub 2019 Aug 18.

Cardiovascular Research Center, Division of Cardiology, Department of Medicine, Harvard Medical School, Massachusetts General Hospital, Charlestown, MA, USA.

Recent works highlight the therapeutic potential of targeting cyclic guanosine monophosphate (cGMP)-dependent pathways in the context of brain ischemia/reperfusion injury (IRI). Although cGMP-dependent protein kinase I (cGKI) has emerged as a key mediator of the protective effects of nitric oxide (NO) and cGMP, the mechanisms by which cGKI attenuates IRI remain poorly understood. We used a novel, conditional cGKI knockout mouse model to study its role in cerebral IRI. We assessed neurological deficit, infarct volume, and cerebral perfusion in tamoxifen-inducible vascular smooth muscle cell-specific cGKI knockout mice and control animals. Stroke experiments revealed greater cerebral infarct volume in smooth muscle cell specific cGKI knockout mice (males: 96 ± 16 mm; females: 93 ± 12 mm, mean±SD) than in all control groups: wild type (males: 66 ± 19; females: 64 ± 14), cGKI control (males: 65 ± 18; females: 62 ± 14), cGKI control with tamoxifen (males: 70 ± 8; females: 68 ± 10). Our results identify, for the first time, a protective role of cGKI in vascular smooth muscle cells during ischemic stroke injury. Moreover, this protective effect of cGKI was found to be independent of gender and was mediated via improved reperfusion. These results suggest that cGKI in vascular smooth muscle cells should be targeted by therapies designed to protect brain tissue against ischemic stroke.
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http://dx.doi.org/10.1177/0271678X19870583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6893979PMC
December 2019

Histone H3K9 Methyltransferase G9a in Oocytes Is Essential for Preimplantation Development but Dispensable for CG Methylation Protection.

Cell Rep 2019 04;27(1):282-293.e4

Division of Epigenomics and Development, Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan. Electronic address:

Mammalian histone methyltransferase G9a (also called EHMT2) deposits H3K9me2 on chromatin and is essential for postimplantation development. However, its role in oogenesis and preimplantation development remains poorly understood. We show that H3K9me2-enriched chromatin domains in mouse oocytes are generally depleted of CG methylation, contrasting with their association in embryonic stem and somatic cells. Oocyte-specific disruption of G9a results in reduced H3K9me2 enrichment and impaired reorganization of heterochromatin in oocytes, but only a modest reduction in CG methylation is detected. Furthermore, in both oocytes and 2-cell embryos, G9a depletion has limited impact on the expression of genes and retrotransposons. Although their CG methylation is minimally affected, preimplantation embryos derived from such oocytes show abnormal chromosome segregation and frequent developmental arrest. Our findings illuminate the functional importance of G9a independent of CG methylation in preimplantation development and call into question the proposed role for H3K9me2 in CG methylation protection in zygotes.
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http://dx.doi.org/10.1016/j.celrep.2019.03.002DOI Listing
April 2019

Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence the outcome of mutagenic DNA repair.

PLoS Biol 2018 12 12;16(12):e2005595. Epub 2018 Dec 12.

MRC Human Genetics Unit, Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, United Kingdom.

Genome editing occurs in the context of chromatin, which is heterogeneous in structure and function across the genome. Chromatin heterogeneity is thought to affect genome editing efficiency, but this has been challenging to quantify due to the presence of confounding variables. Here, we develop a method that exploits the allele-specific chromatin status of imprinted genes in order to address this problem in cycling mouse embryonic stem cells (mESCs). Because maternal and paternal alleles of imprinted genes have identical DNA sequence and are situated in the same nucleus, allele-specific differences in the frequency and spectrum of mutations induced by CRISPR-Cas9 can be unequivocally attributed to epigenetic mechanisms. We found that heterochromatin can impede mutagenesis, but to a degree that depends on other key experimental parameters. Mutagenesis was impeded by up to 7-fold when Cas9 exposure was brief and when intracellular Cas9 expression was low. In contrast, the outcome of mutagenic DNA repair was unaffected by chromatin state, with similar efficiencies of homology-directed repair (HDR) and deletion spectra on maternal and paternal chromosomes. Combined, our data show that heterochromatin imposes a permeable barrier that influences the kinetics, but not the endpoint, of CRISPR-Cas9 genome editing and suggest that therapeutic applications involving low-level Cas9 exposure will be particularly affected by chromatin status.
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http://dx.doi.org/10.1371/journal.pbio.2005595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6306241PMC
December 2018

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

Chronic linaclotide treatment reduces colitis-induced neuroplasticity and reverses persistent bladder dysfunction.

JCI Insight 2018 10 4;3(19). Epub 2018 Oct 4.

Visceral Pain Research Group, Centre for Neuroscience, College of Medicine and Public Health, Flinders University, Bedford Park, South Australia, Australia.

Irritable bowel syndrome (IBS) patients suffer from chronic abdominal pain and extraintestinal comorbidities, including overactive bladder (OAB) and interstitial cystitis/painful bladder syndrome (IC-PBS). Mechanistic understanding of the cause and time course of these comorbid symptoms is lacking, as are clinical treatments. Here, we report that colitis triggers hypersensitivity of colonic afferents, neuroplasticity of spinal cord circuits, and chronic abdominal pain, which persists after inflammation. Subsequently, and in the absence of bladder pathology, colonic hypersensitivity induces persistent hypersensitivity of bladder afferent pathways, resulting in bladder-voiding dysfunction, indicative of OAB/IC-PBS. Daily administration of linaclotide, a guanylate cyclase-C (GC-C) agonist that is restricted to and acts within the gastrointestinal tract, reverses colonic afferent hypersensitivity, reverses neuroplasticity-induced alterations in spinal circuitry, and alleviates chronic abdominal pain in mice. Intriguingly, daily linaclotide administration also reverses persistent bladder afferent hypersensitivity to mechanical and chemical stimuli and restores normal bladder voiding. Linaclotide itself does not inhibit bladder afferents, rather normalization of bladder function by daily linaclotide treatment occurs via indirect inhibition of bladder afferents via reduced nociceptive signaling from the colon. These data support the concepts that cross-organ sensitization underlies the development and maintenance of visceral comorbidities, while pharmaceutical treatments that inhibit colonic afferents may also improve urological symptoms through common sensory pathways.
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http://dx.doi.org/10.1172/jci.insight.121841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6237488PMC
October 2018

GC-B Deficient Mice With Axon Bifurcation Loss Exhibit Compromised Auditory Processing.

Front Neural Circuits 2018 29;12:65. Epub 2018 Aug 29.

Department of Otolaryngology, Head and Neck Surgery, Molecular Physiology of Hearing, Tübingen Hearing Research Centre, University of Tübingen, Tübingen, Germany.

Sensory axon T-like branching (bifurcation) in neurons from dorsal root ganglia and cranial sensory ganglia depends on the molecular signaling cascade involving the secreted factor C-type natriuretic peptide, the natriuretic peptide receptor guanylyl cyclase B (GC-B; also known as Npr2) and cGMP-dependent protein kinase I (cGKI, also known as PKGI). The bifurcation of cranial nerves is suggested to be important for information processing by second-order neurons in the hindbrain or spinal cord. Indeed, mice with a spontaneous GC-B loss of function mutation ( ) display an impaired bifurcation of auditory nerve (AN) fibers. However, these mice did not show any obvious sign of impaired basal hearing. Here, we demonstrate that mice with a targeted inactivation of the GC-B gene (Npr2 , GC-B KO mice) show an elevation of audiometric thresholds. In the inner ear, the cochlear hair cells in GC-B KO mice were nevertheless similar to those from wild type mice, justified by the typical expression of functionally relevant marker proteins. However, efferent cholinergic feedback to inner and outer hair cells was reduced in GC-B KO mice, linked to very likely reduced rapid efferent feedback. Sound-evoked AN responses of GC-B KO mice were elevated, a feature that is known to occur when the efferent axo-dendritic feedback on AN is compromised. Furthermore, late sound-evoked brainstem responses were significantly delayed in GC-B KO mice. This delay in sound response was accompanied by a weaker sensitivity of the auditory steady state response to amplitude-modulated sound stimuli. Finally, the acoustic startle response (ASR) - one of the fastest auditory responses - and the prepulse inhibition of the ASR indicated significant changes in temporal precision of auditory processing. These findings suggest that GC-B-controlled axon bifurcation of spiral ganglion neurons is important for proper activation of second-order neurons in the hindbrain and is a prerequisite for proper temporal auditory processing likely by establishing accurate efferent top-down control circuits. These data hypothesize that the bifurcation pattern of cranial nerves is important to shape spatial and temporal information processing for sensory feedback control.
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http://dx.doi.org/10.3389/fncir.2018.00065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6152484PMC
March 2019

Regulation of the Natriuretic Peptide Receptor 2 (Npr2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is Essential for Bifurcation of Sensory Axons.

J Neurosci 2018 11 24;38(45):9768-9780. Epub 2018 Sep 24.

Max Delbrück Center for Molecular Medicine, 13092 Berlin, Germany,

cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase Npr2 (also known as guanylyl cyclase B) by the ligand CNP controls sensory axon bifurcation of DRG and cranial sensory ganglion (CSG) neurons entering the spinal cord or hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine and threonine residues in its kinase homology domain (KHD). However, it is unknown whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD of Npr2 were substituted by alanine (Npr2-7A), resulting in a nonphosphorylatable enzyme. Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the absence of CNP-induced cGMP generation in the mutant. Consequently, bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of all of the seven serine and threonine sites by glutamic acid (Npr2-7E). Furthermore, we demonstrate that the mutation causes dwarfism as described for global Npr2 mutants. In conclusion, our studies provide strong evidence that phosphorylation of the seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of Npr2-mediated cGMP signaling which affects physiological processes, such as axon bifurcation and bone growth. The branching of axons is a morphological hallmark of virtually all neurons. It allows an individual neuron to innervate different targets and to communicate with neurons located in different regions of the nervous system. The natriuretic peptide receptor 2 (Npr2), a transmembrane guanylyl cyclase, is essential for the initiation of bifurcation of sensory axons when entering the spinal cord or the hindbrain. By using two genetically engineered mouse lines, we show that phosphorylation of specific serine and threonine residues in juxtamembrane regions of Npr2 are required for its enzymatic activity and for axon bifurcation. These investigations might help to understand the regulation of Npr2 and its integration in intracellular signaling systems.
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http://dx.doi.org/10.1523/JNEUROSCI.0495-18.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6222061PMC
November 2018

Parallels between Mammalian Mechanisms of Monoallelic Gene Expression.

Trends Genet 2018 12 11;34(12):954-971. Epub 2018 Sep 11.

Institute of Molecular Genetics of Montpellier (IGMM), CNRS and the University of Montpellier, 1919 route de Mende, 34293 Montpellier, France. Electronic address:

Different types of monoallelic gene expression are present in mammals, some of which are highly flexible, whereas others are more rigid. These include allelic exclusion at antigen receptor loci, the expression of olfactory receptor genes, genomic imprinting, X-chromosome inactivation, and random monoallelic expression (MAE). Although these processes play diverse biological roles, and arose through different selective pressures, the underlying epigenetic mechanisms show striking resemblances. Regulatory transcriptional events are important in all systems, particularly in the specification of MAE. Combined with comparative studies between species, this suggests that the different MAE systems found in mammals may have evolved from analogous ancestral processes.
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http://dx.doi.org/10.1016/j.tig.2018.08.005DOI Listing
December 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 Imaging in Brain Slices Reveals Brain Region-Specific Activity of NO-Sensitive Guanylyl Cyclases (NO-GCs) and NO-GC Stimulators.

Int J Mol Sci 2018 Aug 7;19(8). Epub 2018 Aug 7.

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

Impaired NO-cGMP signaling has been linked to several neurological disorders. NO-sensitive guanylyl cyclase (NO-GC), of which two isoforms-NO-GC1 and NO-GC2-are known, represents a promising drug target to increase cGMP in the brain. Drug-like small molecules have been discovered that work synergistically with NO to stimulate NO-GC activity. However, the effects of NO-GC stimulators in the brain are not well understood. In the present study, we used Förster/fluorescence resonance energy transfer (FRET)-based real-time imaging of cGMP in acute brain slices and primary neurons of cGMP sensor mice to comparatively assess the activity of two structurally different NO-GC stimulators, IWP-051 and BAY 41-2272, in the cerebellum, striatum and hippocampus. BAY 41-2272 potentiated an elevation of cGMP induced by the NO donor DEA/NO in all tested brain regions. Interestingly, IWP-051 potentiated DEA/NO-induced cGMP increases in the cerebellum and striatum, but not in the hippocampal CA1 area or primary hippocampal neurons. The brain-region-selective activity of IWP-051 suggested that it might act in a NO-GC isoform-selective manner. Results of mRNA in situ hybridization indicated that the cerebellum and striatum express NO-GC1 and NO-GC2, while the hippocampal CA1 area expresses mainly NO-GC2. IWP-051-potentiated DEA/NO-induced cGMP signals in the striatum of NO-GC2 knockout mice but was ineffective in the striatum of NO-GC1 knockout mice. These results indicate that IWP-051 preferentially stimulates NO-GC1 signaling in brain slices. Interestingly, no evidence for an isoform-specific effect of IWP-051 was observed when the cGMP-forming activity of whole brain homogenates was measured. This apparent discrepancy suggests that the method and conditions of cGMP measurement can influence results with NO-GC stimulators. Nevertheless, it is clear that NO-GC stimulators enhance cGMP signaling in the brain and should be further developed for the treatment of neurological diseases.
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http://dx.doi.org/10.3390/ijms19082313DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122017PMC
August 2018

Real-Time Imaging Reveals Augmentation of Glutamate-Induced Ca Transients by the NO-cGMP Pathway in Cerebellar Granule Neurons.

Int J Mol Sci 2018 Jul 26;19(8). Epub 2018 Jul 26.

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

Dysfunctions of NO-cGMP signaling have been implicated in various neurological disorders. We have studied the potential crosstalk of cGMP and Ca signaling in cerebellar granule neurons (CGNs) by simultaneous real-time imaging of these second messengers in living cells. The NO donor DEA/NO evoked cGMP signals in the granule cell layer of acute cerebellar slices from transgenic mice expressing a cGMP sensor protein. cGMP and Ca dynamics were visualized in individual CGNs in primary cultures prepared from 7-day-old cGMP sensor mice. DEA/NO increased the intracellular cGMP concentration and augmented glutamate-induced Ca transients. These effects of DEA/NO were absent in CGNs isolated from knockout mice lacking NO-sensitive guanylyl cyclase. Furthermore, application of the cGMP analogues 8-Br-cGMP and 8-pCPT-cGMP, which activate cGMP effector proteins such as cyclic nucleotide-gated cation channels and cGMP-dependent protein kinases (cGKs), also potentiated glutamate-induced Ca transients. Western blot analysis failed to detect cGK type I or II in our primary CGNs. The addition of phosphodiesterase (PDE) inhibitors during cGMP imaging showed that CGNs degrade cGMP mainly via Zaprinast-sensitive PDEs, most likely PDE5 and/or PDE10, but not via PDE1, 2, or 3. In sum, these data delineate a cGK-independent NO-cGMP signaling cascade that increases glutamate-induced Ca signaling in CGNs. This cGMP⁻Ca crosstalk likely affects neurotransmitter-stimulated functions of CGNs.
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http://dx.doi.org/10.3390/ijms19082185DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6121606PMC
July 2018

Environmental effects on chromatin repression at imprinted genes and endogenous retroviruses.

Curr Opin Chem Biol 2018 08 23;45:139-147. Epub 2018 May 23.

Institute of Molecular Genetics of Montpellier (IGMM), CNRS and University of Montpellier, 1919, route de Mende, 34293 Montpellier, France. Electronic address:

Environmental factors can perturb epigenetic regulation. In mammals, most studies have focused on repressive DNA methylation. Two attractive model systems to monitor environmentally triggered drifts in DNA methylation are genomic imprinting and endogenous retroviruses (ERVs), particularly intracisternal-A particles (IAPs). These systems show mechanistic similarities in their repressive chromatin organization, which in somatic cells is comparable between the DNA-methylated alleles of imprinted differentially methylated regions (DMRs) and repressed ERVs. Here, we present how during development, nutrition and chemical components can perturb DNA methylation at imprinted genes and ERVs, and discuss the still poorly understood underlying mechanisms.
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http://dx.doi.org/10.1016/j.cbpa.2018.04.015DOI Listing
August 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
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