Max Planck Institute For Herz Und Lungenforschung

Bad Nauheim, Germany

Max Planck Institute For Herz Und Lungenforschung

Bad Nauheim, Germany
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Panza P.,Max Planck Institute For Entwicklungsbiologie | Sitko A.A.,Columbia University | Maischein H.-M.,Max Planck Institute For Entwicklungsbiologie | Maischein H.-M.,Max Planck Institute For Herz Und Lungenforschung | And 6 more authors.
Neural Development | Year: 2015

Background: In the visual system of most binocular vertebrates, the axons of retinal ganglion cells (RGCs) diverge at the diencephalic midline and extend to targets on both ipsi- and contralateral sides of the brain. While a molecular mechanism explaining ipsilateral guidance decisions has been characterized, less is known of how RGC axons cross the midline. Results: Here, we took advantage of the zebrafish, in which all RGC axons project contralaterally at the optic chiasm, to characterize Islr2 as an RGC receptor required for complete retinal axon midline crossing. We used a systematic extracellular protein-protein interaction screening assay to identify two Vasorin paralogs, Vasna and Vasnb, as specific Islr2 ligands. Antibodies against Vasna and Vasnb reveal cellular populations surrounding the retinal axon pathway, suggesting the involvement of these proteins in guidance decisions made by axons of the optic nerve. Specifically, Vasnb marks the membranes of a cellular barricade located anteriorly to the optic chiasm, a structure termed the "glial knot" in higher vertebrates. Loss of function mutations in either vasorin paralog, individually or combined, however, do not exhibit an overt retinal axon projection phenotype, suggesting that additional midline factors, acting either independently or redundantly, compensate for their loss. Analysis of Islr2 knockout mice supports a scenario in which Islr2 controls the coherence of RGC axons through the ventral midline and optic tract. Conclusions: Although stereotypic guidance of RGC axons at the vertebrate optic chiasm is controlled by multiple, redundant mechanisms, and despite the differences in ventral diencephalic tissue architecture, we identify a novel role for the LRR receptor Islr2 in ensuring proper axon navigation at the optic chiasm of both zebrafish and mouse. © 2015 Panza et al.

Weissmann N.,Justus Liebig University | Sydykov A.,Justus Liebig University | Kalwa H.,University of Marburg | Kalwa H.,Harvard University | And 20 more authors.
Nature Communications | Year: 2012

Lung ischaemia-reperfusion-induced oedema (LIRE) is a life-threatening condition that causes pulmonary oedema induced by endothelial dysfunction. Here we show that lungs from mice lacking nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (Nox2y/-) or the classical transient receptor potential channel 6 (TRPC6-/-) are protected from LIR-induced oedema (LIRE). Generation of chimeric mice by bone marrow cell transplantation and endothelial-specific Nox2 deletion showed that endothelial Nox2, but not leukocytic Nox2 or TRPC6, are responsible for LIRE. Lung endothelial cells from Nox2- or TRPC6-deficient mice showed attenuated ischaemia-induced Ca 2+ influx, cellular shape changes and impaired barrier function. Production of reactive oxygen species was completely abolished in Nox2 y/- cells. A novel mechanistic model comprising endothelial Nox2-derived production of superoxide, activation of phospholipase C-γ, inhibition of diacylglycerol (DAG) kinase, DAG-mediated activation of TRPC6 and ensuing LIRE is supported by pharmacological and molecular evidence. This mechanism highlights novel pharmacological targets for the treatment of LIRE. © 2012 Macmillan Publishers Limited. All rights reserved.

Oberoi-Khanuja T.K.,Goethe University Frankfurt | Karreman C.,University of Konstanz | Larisch S.,Haifa University | Rapp U.R.,Max Planck Institute For Herz Und Lungenforschung | Rajalingam K.,Goethe University Frankfurt
Journal of Biological Chemistry | Year: 2012

Inhibitor of apoptosis (IAPs) proteins are characterized by the presence of evolutionarily conserved baculoviral inhibitor of apoptosis repeat (BIR) domains, predominantly known for their role in inhibiting caspases and, thereby, apoptosis. We have shown previously that multi-BIR domain-containing IAPs, cellular IAPs, and X-linked IAP can control tumor cell migration by directly regulating the protein stability of C-RAF kinase. Here, we extend our observations to a single BIR domain containing IAP family member melanoma-IAP (ML-IAP). We show that ML-IAP can directly bind to C-RAF and that ML-IAP depletion leads to an increase in C-RAF protein levels, MAPK activation, and cell migration in melanoma cells. Thus, our results unveil a thus far unknown role for ML-IAP in controlling C-RAF stability and cell migration. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.

Michaelis U.R.,Goethe University Frankfurt | Michaelis U.R.,German Center for Cardiovascular Research | Chavakis E.,Goethe University Frankfurt | Chavakis E.,German Center for Cardiovascular Research | And 19 more authors.
Circulation Research | Year: 2013

RATIONALE:: Polarity proteins are involved in the apico-basal orientation of epithelial cells, but relatively little is known regarding their function in mesenchymal cells. OBJECTIVE:: We hypothesized that polarity proteins also contribute to endothelial processes like angiogenesis. METHODS AND RESULTS:: Screening of endothelial cells revealed high expression of the polarity protein Scribble (Scrib). On fibronectin-coated carriers Scrib siRNA (siScrib) blocked directed but not random migration of human umbilical vein endothelial cells and led to an increased number and disturbed orientation of cellular lamellipodia. Coimmunoprecipitation/mass spectrometry and glutathione S-transferase (GST) pulldown assays identified integrin α5 as a novel Scrib interacting protein. By total internal reflection fluorescence (TIRF) microscopy, Scrib and integrin α5 colocalize at the basal plasma membrane of endothelial cells. Western blot and fluorescence activated cell sorting (FACS) analysis revealed that silencing of Scrib reduced the protein amount and surface expression of integrin α5 whereas surface expression of integrin αV was unaffected. Moreover, in contrast to fibronectin, the ligand of integrin α5, directional migration on collagen mediated by collagen-binding integrins was unaffected by siScrib. Mechanistically, Scrib supported integrin α5 recycling and protein stability by blocking its interaction with Rab7a, its translocation into lysosomes, and its subsequent degradation by pepstatin-sensitive proteases. In siScrib-treated cells, reinduction of the wild-type protein but not of PSD95, Dlg, ZO-1 (PDZ), or leucine rich repeat domain mutants restored integrin α5 abundance and directional cell migration. The downregulation of Scrib function in Tg(kdrl:EGFP) transgenic zebrafish embryos delayed the angiogenesis of intersegmental vessels. CONCLUSIONS:: Scrib is a novel regulator of integrin α5 turnover and sorting, which is required for oriented cell migration and sprouting angiogenesis. © 2013 American Heart Association, Inc.

Schafer A.,German Cancer Research Center | Schomacher L.,German Cancer Research Center | Barreto G.,German Cancer Research Center | Barreto G.,Max Planck Institute For Herz Und Lungenforschung | And 3 more authors.
PLoS ONE | Year: 2010

Gemcitabine is a cytotoxic cytidine analog, which is widely used in anti-cancer therapy. One mechanism by which gemcitabine acts is by inhibiting nucleotide excision repair (NER). Recently NER was implicated in Gadd45 mediated DNA demethylation and epigenetic gene activation. Here we analyzed the effect of gemcitabine on DNA demethylation. We find that gemcitabine inhibits specifically Gadd45a mediated reporter gene activation and DNA demethylation, similar to the topoisomerase I inhibitor camptothecin, which also inhibits NER. In contrast, base excision repair inhibitors had no effect on DNA demethylation. In Xenopus oocytes, gemcitabine inhibits DNA repair synthesis accompanying demethylation of oct4. n mammalian cells, gemcitabine induces DNA hypermethylation and silencing of MLH1. The results indicate that gemcitabine induces epigenetic gene silencing by inhibiting repair mediated DNA demethylation. Thus, gemcitabine can function epigenetically and provides a tool to manipulate DNA methylation. © 2010 Schäfer et al.

Wystub K.,Max Planck Institute For Herz Und Lungenforschung | Besser J.,Max Planck Institute For Herz Und Lungenforschung | Bachmann A.,Max Planck Institute For Herz Und Lungenforschung | Boettger T.,Max Planck Institute For Herz Und Lungenforschung | Braun T.,Max Planck Institute For Herz Und Lungenforschung
PLoS Genetics | Year: 2013

miRNAs are small RNAs directing many developmental processes by posttranscriptional regulation of protein-coding genes. We uncovered a new role for miR-1-1/133a-2 and miR-1-2/133a-1 clusters in the specification of embryonic cardiomyocytes allowing transition from an immature state characterized by expression of smooth muscle (SM) genes to a more mature fetal phenotype. Concomitant knockout of miR-1-1/133a-2 and miR-1-2/133a-1 released suppression of the transcriptional co-activator myocardin, a major regulator of SM gene expression, but not of its binding partner SRF. Overexpression of myocardin in the embryonic heart essentially recapitulated the miR-1/133a mutant phenotype at the molecular level, arresting embryonic cardiomyocytes in an immature state. Interestingly, the majority of postulated miR-1/133a targets was not altered in double mutant mice, indicating that the ability of miR-1/133a to suppress target molecules strongly depends on the cellular context. Finally, we show that myocardin positively regulates expression of miR-1/133a, thus constituting a negative feedback loop that is essential for early cardiac development. © 2013 Wystub et al.

Looso M.,Max Planck Institute For Herz Und Lungenforschung | Borchardt T.,Max Planck Institute For Herz Und Lungenforschung | Kruger M.,Max Planck Institute For Herz Und Lungenforschung | Braun T.,Max Planck Institute For Herz Und Lungenforschung
Molecular and Cellular Proteomics | Year: 2010

Despite progress in the characterization of their genomes, proteomes of several model organisms are often only poorly characterized. This problem is aggravated by the presence of large numbers of expressed sequence tag clones that lack homologues in other species, which makes it difficult to identify new proteins irrespective of whether such molecules are involved in species-specific biological processes. We have used a pulsed stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry method, which is based on the detection of paired peptides after [13C6]lysine incorporation into proteins in vivo, to greatly increase the confidence of protein identification in cross-species database searches. The method was applied to identify nearly 3000 proteins in regenerating tails of the urodele amphibian Notophthalmus viridescens, which possesses outstanding capabilities in the regeneration of complex tissues. We reason that pulsed in vivo SILAC represents a versatile tool to identify new proteins in species for which only limited sequence information exists. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

Wilhelm K.,Max Planck Institute For Herz Und Lungenforschung | Potente M.,Max Planck Institute For Herz Und Lungenforschung
BioSpektrum | Year: 2016

Recent insights into fundamental aspects of angiogenesis move meta-bolism into the spotlight and highlight the importance of endothelial metabolic adaptation for vascular growth. This notion stems from the realization that endothelial cells (ECs) have to meet the metabolic demands of migration and proliferation when they switch from quiescence to vessel growth. Here we discuss how ECs adapt their metabolism during the angiogenic switch and how the forkhead transcription factor FOXO1 regulates this process. © 2016, Springer-Verlag Berlin Heidelberg.

Kohlstedt K.,Goethe University Frankfurt | Trouvain C.,Goethe University Frankfurt | Boettger T.,Max Planck Institute For Herz Und Lungenforschung | Shi L.,Goethe University Frankfurt | And 2 more authors.
Circulation Research | Year: 2013

RATIONALE: High-angiotensin-converting enzyme (ACE)-levels are associated with cardiovascular disease, but little is known about the regulation of its expression. OBJECTIVE: To assess the molecular mechanisms regulating endothelial ACE expression focusing on the role of the AMP-activated protein kinase (AMPK) and miR-143/145. METHODS AND RESULTS: Shear stress decreased ACE expression in cultured endothelial cells, an effect prevented by downregulating AMPKα2 but not AMPKα1. AMPKα2-/- mice expressed higher ACE levels than wild-type littermates resulting in impaired hindlimb vasodilatation to the ACE substrate, bradykinin. The latter response was also evident in animals lacking the AMPKα2 subunit only in endothelial cells. In cultured endothelial cells, miR-143/145 levels were increased by shear stress in an AMPKα2-dependent manner, and miR-143/145 overexpression decreased ACE expression. The effect of shear stress was unrelated to an increase in miR-143/145 promoter activity and transcription but could be attributed to post-transcriptional regulation of precursor-miR-143/145 by AMPKα2. The AMPK substrate, p53, can enhance the post-transcriptional processing of several microRNAs, including miR-143/145. We found that shear stress elicited the AMPKα2-dependent phosphorylation of p53 (on Ser15), and that p53 downregulation prevented the shear stress-induced decrease in ACE expression. Streptozotocin-induced diabetes mellitus in mice was studied as a pathophysiological model of altered AMPK activity. Diabetes mellitus increased tissue phosphorylation of the AMPK substrates, p53 and acetyl-coenzyme A carboxylase, changes that correlated with increased miR-143/145 levels and decreased ACE expression. CONCLUSIONS: AMPKα2 suppresses endothelial ACE expression via the phosphorylation of p53 and upregulation of miR-143/145. Post-transcriptional regulation of miR-143/145 may contribute to the vascular complications associated with diabetes mellitus. Copyright © 2013 American Heart Association, Inc.

Groger N.,Max Planck Institute For Herz Und Lungenforschung | Frohlich H.,Max Planck Institute For Herz Und Lungenforschung | Frohlich H.,University of Tübingen | Maier H.,Leibniz University of Hanover | And 4 more authors.
Journal of Biological Chemistry | Year: 2010

Maintenance of ion concentration gradients is essential for the function of many organs, including the kidney, the cornea, and the inner ear. Ion concentrations and fluid content in the cornea are regulated by endothelial cells that separate the collagenous avascular corneal stroma from the anterior eye chamber. Failure to maintain correct ion concentrations leads to swelling and destruction of the cornea. In the inner ear, the stria vascularis is responsible for generating proper ion concentrations in the endolymph, which is essential for hearing. Mutations of SLC4A11 in humans lead to syndromes associated with corneal dystrophy and perceptive deafness. The molecular mechanisms underlying these symptoms are poorly understood, impeding therapeutic interventions. The ion transporter SLC4A11 mediates sodium-dependent transport of borate as well as flux of sodium and hydroxyl ions in vitro. Here, we show that SLC4A11 is expressed in the endothelial cells of the cornea where it prevents severe morphological changes of the cornea caused by increased sodium chloride concentrations in the stroma. In the inner ear, SLC4A11 is located in fibrocytes underlying the stria vascularis. Loss of SLC4A11 leads to morphological changes in the fibrocytes and deafness. Wedemonstrate that SLC4A11 is essential for the generation of the endocochlear potential but not for regulation of potassium concentrations in the endolymph. In the kidney, SLC4A11 is expressed in the thin descending limb of Henle loop. SLC4A11 is essential for urinary concentration, suggesting that SLC4A11 participates in the countercurrent multiplication that concentrates urine in the kidney medulla. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.

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