Jungmann O.,University of Munster |
Nikolovska K.,University of Munster |
Stock C.,University of Munster |
Schulz J.-N.,University of Cologne |
And 5 more authors.
PLoS ONE | Year: 2012
Decorin, a small leucine-rich proteoglycan harboring a dermatan sulfate chain at its N-terminus, is involved in regulating matrix organization and cell signaling. Loss of the dermatan sulfate of decorin leads to an Ehlers-Danlos syndrome characterized by delayed wound healing. Decorin-null (Dcn-/-) mice display a phenotype similar to that of EDS patients. The fibrillar collagen phenotype of Dcn-/- mice could be rescued in vitro by decorin but not with decorin lacking the glycosaminoglycan chain. We utilized a 3D cell culture model to investigate the impact of the altered extracellular matrix on Dcn-/- fibroblasts. Using 2D gel electrophoresis followed by mass spectrometry, we identified vimentin as one of the proteins that was differentially upregulated by the presence of decorin. We discovered that a decorin-deficient matrix leads to abnormal nuclear morphology in the Dcn-/- fibroblasts. This phenotype could be rescued by the decorin proteoglycan but less efficiently by the decorin protein core. Decorin treatment led to a significant reduction of the α2β1 integrin at day 6 in Dcn-/- fibroblasts, whereas the protein core had no effect on β1. Interestingly, only the decorin core induced mRNA synthesis, phosphorylation and de novo synthesis of vimentin indicating that the proteoglycan decorin in the extracellular matrix stabilizes the vimentin intermediate filament system. We could support these results in vivo, because the dermis of wild-type mice have more vimentin and less β1 integrin compared to Dcn-/-. Furthermore, the α2β1 null fibroblasts also showed a reduced amount of vimentin compared to wild-type. These data show for the first time that decorin has an impact on the biology of α2β1 integrin and the vimentin intermediate filament system. Moreover, our findings provide a mechanistic explanation for the reported defects in wound healing associated with the Dcn-/- phenotype. © 2012 Jungmann et al.
PubMed | Serend ip GmbH, University of Würzburg and University of Heidelberg
Type: | Journal: Frontiers in cellular neuroscience | Year: 2014
Stabilization of the blood-brain barrier during and after stroke can lead to less adverse outcome. For elucidation of underlying mechanisms and development of novel therapeutic strategies validated in vitro disease models of the blood-brain barrier could be very helpful. To mimic in vitro stroke conditions we have established a blood-brain barrier in vitro model based on mouse cell line cerebEND and applied oxygen/glucose deprivation (OGD). The role of astrocytes in this disease model was investigated by using cell line C6. Transwell studies pointed out that addition of astrocytes during OGD increased the barrier damage significantly in comparison to the endothelial monoculture shown by changes of transendothelial electrical resistance as well as fluorescein permeability data. Analysis on mRNA and protein levels by qPCR, western blotting and immunofluorescence microscopy of tight junction molecules claudin-3,-5,-12, occludin and ZO-1 revealed that their regulation and localisation is associated with the functional barrier breakdown. Furthermore, soluble factors of astrocytes, OGD and their combination were able to induce changes of functionality and expression of ABC-transporters Abcb1a (P-gp), Abcg2 (bcrp), and Abcc4 (mrp4). Moreover, the expression of proteases (matrixmetalloproteinases MMP-2, MMP-3, MMP-9, and t-PA) as well as of their endogenous inhibitors (TIMP-1, TIMP-3, PAI-1) was altered by astrocyte factors and OGD which resulted in significant changes of total MMP and t-PA activity. Morphological rearrangements induced by OGD and treatment with astrocyte factors were confirmed at a nanometer scale using atomic force microscopy. In conclusion, astrocytes play a major role in blood-brain barrier breakdown during OGD in vitro.
Thoelking G.,University of Munster |
Reiss B.,University of Munster |
Reiss B.,University of Regensburg |
Wegener J.,University of Regensburg |
And 4 more authors.
Nanotechnology | Year: 2010
Inflammation and cellular fibrosis often imply an involvement of the cytokine TGF-β1. TGF-β1 induces epithelial-to-mesenchymal transdifferentiation (EMT), a term describing the loss of epithelium-specific function. Indicative for this process are an elongated cell shape parallel to stress fibre formation. Many signalling pathways of TGF-β1 have been discovered, but mechanical aspects have not yet been investigated. In this study, atomic force microscopy (AFM) was used to analyse surface topography and mechanical properties of EMT in proximal kidney tubule epithelium (NRK52E). Elongated cells, an increase of stress fibre formation and a loss of microvillus compatible structures were observed as characteristic signs of EMT. Furthermore, AFM could identify an increase in stiffness by 71% after six days of stimulation with TGF-β1. As a novel topographical phenomenon, nodular protrusions emerged at the cell-cell junctions. They occurred preferentially at sites where stress fibres cross the border. Since these nodular protrusions were sensitive to inhibitors of force generation, they can indicate intracellular tension. The results demonstrate a manifest impact of elevated tension on the cellular topography. © 2010 IOP Publishing Ltd.
Agency: European Commission | Branch: H2020 | Program: MSCA-RISE | Phase: MSCA-RISE-2014 | Award Amount: 567.00K | Year: 2015
Cancer is a leading cause of mortality within the aging European population. Therapeutic targeting is hampered by the complexity of the disease, which includes not only molecular changes within the tumor cell itself, but also within its microenvironment. Tumor angiogenesis, tumor-stroma interactions, interactions with immune cells, with the extracellular matrix and cancer stem cell niches allow for malignant cell survival and promote metastasis, the leading cause for cancer-associated mortality. Proteoglycans (PGs) and glycosaminoglycans (GAGs) structurally diverse carbohydrates of the extracellular matrix and cell surfaces - have emerged as novel biomarkers and molecular players both within tumor cells and their microenvironment, as they integrate signals from growth factors, chemokines and integrins, and cell-cell as well as matrix adhesion. Importantly, their expression is dysregulated in numerous tumor entities, and has been shown to modulate each of the hallmarks of cancer as defined by Hanahan and Weinberg (Cell 2011). We hypothesize that dysregulated function of PGs and GAGs simultaneously affects all molecular steps towards cancer metastasis as a general principle applicable to multiple tumor entities. Pharmacological modulation of their function thus emerges as an attractive multitargeted antitumoral approach which simultaneously acts at multiple levels of disease progression. Besides providing extensive knowledge transfer and training for researchers, the combined expertise of the GLYCANC consortium aims at performing a detailed structural analysis of PG and GAG glycans in disease using state-of-the art methodology, analysing their regulation via epigenetic mechanisms and microRNAs, and elucidating molecular mechanisms underlying aberrant PG and GAG function. GLYCANC will lead to a deeper understanding of glycan structures and glycan-dependent mechanisms promoting cancer progression, providing the basis for rational multitargeted anticancer approaches.
Isac L.,University of Munster |
Isac L.,University of Bucharest |
Thoelking G.,University of Munster |
Schwab A.,University of Munster |
And 3 more authors.
Analytical and Bioanalytical Chemistry | Year: 2011
A demanding task of medicine is to understand and control the immune system. Central players in the cellular immune response are the leukocytes that leave the blood stream for host defense. Endothelial cells limit the emigration rate of leukocytes. Being located between blood and tissues, they permit or deny the passage. The exact mechanism of this process called diapedesis is not solved yet. Leukocytes can principally traverse either between cells (paracellularly) or directly through an individual endothelial cell (transcellularly). The transcellular way has recently gained experimental support, but it is not clear how the endothelial cytoskeleton manages to open and close a transmigratory channel. Atomic force microscopy was used to investigate the endothelial cytoskeleton. In order to directly access the leukocyte-endothelial interaction site, we applied a special protocol ("nanosurgery"). As a result, the endothelial cell turned out to become softer in a confined region strictly underneath the leukocyte. Fluorescence microscopy confirmed a depolymerization of the f-actin strands at the invasion site. Leukocytes dramatically rearrange the endothelial cytoskeleton to form transmigratory channels. © 2010 Springer-Verlag.
Agency: European Commission | Branch: H2020 | Program: MSCA-RISE | Phase: MSCA-RISE-2015 | Award Amount: 346.50K | Year: 2016
Endometriosis (the presence of endometrial-like tissue or lesions outside the uterus) is a chronic inflammatory disease affecting 6-10% of reproductive age women. It is associated with pelvic pain, painful periods, pain with sexual intercourse, and subfertility. The socioeconomic impacts of endometriosis are considerable. The recent WERF EndoCost study (a worldwide cost-of-illness study) reported that endometriosis reduced work productivity in 51% and negatively affected relationships in 50% of women with endometriosis. Current treatment options include invasive and repeated surgeries, or hormonal suppression with significant side effects analogous to a premature menopause. While several general concepts of the etiology of the disease such as retrograde menstruation, coelomic metaplasia, lymphovascular metastasis or the embryonic rest theory have been developed, the molecular mechanisms underlying the disease are currently still incompletely understood, hampering the development of efficient targeted therapies with limited side effects. MOMENDO utilizes the potential of an interdisciplinary consortium of leading world endometriosis experts to provide a deeper understanding of key molecular processes contributing to disease etiology and progression. By employing a wide range of experimental methods (biomarker and epigenetic studies on patient tissues, advanced animal models of the disease, proprietary atomic force microscopy for marker-free diagnostics) and novel and innovative conceptual approaches (adult stem cells, microRNAs, iron-induced inflammatory responses, novel endocrine concepts), MOMENDO will substantially contribute to a deeper understanding of the molecular mechanisms that explain the inflammatory pain associated with endometriosis and the persistent growth of endometriosis lesions. By combining the respective strengths of non-academic and clinical partners, MOMENDO expect to successfully translate these findings into novel therapeutic approaches.
Riethmuller C.,Serend IP GmbH |
McAleer M.A.,Our Ladys Childrens Hospital |
Koppes S.A.,Coronel Institute of Occupational Health |
Abdayem R.,University of Lyon |
And 9 more authors.
Journal of Allergy and Clinical Immunology | Year: 2015
Background Loss-of-function (LOF) mutations in the filaggrin gene (FLG) are a well-replicated risk factor for atopic dermatitis (AD) and are known to cause an epidermal barrier defect. The nature of this barrier defect is not fully understood. Patients with AD with FLG LOF mutations are known to have more persistent disease, more severe disease, and greater risk of food allergies and eczema herpeticum. Abnormalities in corneocyte morphology have been observed in patients with AD, including prominent villus-like projections (VP); however, these ultrastructural features have not been systematically studied in patients with AD in relation to FLG genotype and acute and convalescent status. Objective We sought to quantitatively explore the relationship between FLG genotype, filaggrin breakdown products (natural moisturizing factor [NMF]), and corneocyte morphology in patients with AD. Methods We studied 15 children at first presentation of AD and after 6 weeks of standard therapy. We applied atomic force microscopy to study corneocyte conformation in patients with AD stratified by FLG status and NMF level. By using a new quantitative methodology, the number of VPs per investigated corneocyte area was assessed and expressed as the Dermal Texture Index score. Corneocytes were also labeled with an anti-corneodesmosin antibody and visualized with scanning electron microscopy. Results We found a strong correlation between NMF levels and Dermal Texture Index scores in both acute and convalescent states (respective r = -0.80 and -0.75, P <.001 and P =.002). Most, but not all, VPs showed the presence of corneodesmosin abundantly all over the cell surface in homozygous/compound heterozygous FLG patients and, to a lesser extent, in heterozygous and wild-type patients. Conclusions NMF levels are highly correlated with corneocyte morphology in patients with AD. These corneocyte conformational changes shed further insight into the filaggrin-deficient phenotype and help explain the barrier defect in patients with AD with FLG LOF mutations. © 2015 The Authors.
PubMed | University of Amsterdam, Serend ip GmbH, Copenhagen University and University of Munster
Type: Journal Article | Journal: Skin research and technology : official journal of International Society for Bioengineering and the Skin (ISBS) [and] International Society for Digital Imaging of Skin (ISDIS) [and] International Society for Skin Imaging (ISSI) | Year: 2016
The skin barrier protects the organism against exogenous stressors and simultaneously prevents excessive water loss. While the delicate regulation of skin barrier is not completely understood, morphological and histological evaluation remain key features of clinical investigations. Here, we extended the phenotypic perspective down to the nanoscale.Corneocyte samples were obtained non-invasively by a standard tape stripping procedure from 21 indviduals. Scanning electron (SEM) and atomic force microcopy (AFM) were used to record nanoscale topography. Circular nano-objects were identified and these were quantitated through computer vision.Typical dimensions of 273nm height and 305nm width. We showed that their density does not correlate to age or pigmentation in healthy subjects, but that they were clearly elevated in corneocytes from patients with atopic dermatitis, a common inflammatory skin condition.The presence of these corneocyte-nanostructures might be used as a diagnostic parameter for skin disorders - even in cases below a clinical threshold.
PubMed | Serend ip GmbH, University of Lyon, Trinity College Dublin, Coronel Institute of Occupational Health and 2 more.
Type: Journal Article | Journal: The Journal of allergy and clinical immunology | Year: 2015
Loss-of-function (LOF) mutations in the filaggrin gene (FLG) are a well-replicated risk factor for atopic dermatitis (AD) and are known to cause an epidermal barrier defect. The nature of this barrier defect is not fully understood. Patients with AD with FLG LOF mutations are known to have more persistent disease, more severe disease, and greater risk of food allergies and eczema herpeticum. Abnormalities in corneocyte morphology have been observed in patients with AD, including prominent villus-like projections (VP); however, these ultrastructural features have not been systematically studied in patients with AD in relation to FLG genotype and acute and convalescent status.We sought to quantitatively explore the relationship between FLG genotype, filaggrin breakdown products (natural moisturizing factor [NMF]), and corneocyte morphology in patients with AD.We studied 15 children at first presentation of AD and after 6 weeks of standard therapy. We applied atomic force microscopy to study corneocyte conformation in patients with AD stratified by FLG status and NMF level. By using a new quantitative methodology, the number of VPs per investigated corneocyte area was assessed and expressed as the Dermal Texture Index score. Corneocytes were also labeled with an anti-corneodesmosin antibody and visualized with scanning electron microscopy.We found a strong correlation between NMF levels and Dermal Texture Index scores in both acute and convalescent states (respective r = -0.80 and -0.75, P < .001 and P = .002). Most, but not all, VPs showed the presence of corneodesmosin abundantly all over the cell surface in homozygous/compound heterozygous FLG patients and, to a lesser extent, in heterozygous and wild-type patients.NMF levels are highly correlated with corneocyte morphology in patients with AD. These corneocyte conformational changes shed further insight into the filaggrin-deficient phenotype and help explain the barrier defect in patients with AD with FLG LOF mutations.
PubMed | Institute associated Research Group Cell adhesion and cell polarity, Hannover Medical School, Serend ip GmbH and University of Munster
Type: Journal Article | Journal: PloS one | Year: 2016
Endothelial barriers have a central role in inflammation as they allow or deny the passage of leukocytes from the vasculature into the tissue. To bind leukocytes, endothelial cells form adhesive clusters containing tetraspanins and ICAM-1, so-called endothelial adhesive platforms (EAPs). Upon leukocyte binding, EAPs evolve into docking structures that emanate from the endothelial surface while engulfing the leukocyte. Here, we show that TNF- is sufficient to induce apical protrusions in the absence of leukocytes. Using advanced quantitation of atomic force microscopy (AFM) recordings, we found these structures to protrude by 160 80 nm above endothelial surface level. Confocal immunofluorescence microscopy proved them positive for ICAM-1, JAM-A, tetraspanin CD9 and f-actin. Microvilli formation was inhibited in the absence of CD9. Our findings indicate that stimulation with TNF- induces nanoscale changes in endothelial surface architecture and that--via a tetraspanin CD9 depending mechanism--the EAPs rise above the surface to facilitate leukocyte capture.