Publications by authors named "Bent Brachvogel"

63 Publications

Mitochondrial respiratory chain function promotes extracellular matrix integrity in cartilage.

J Biol Chem 2021 Sep 22;297(4):101224. Epub 2021 Sep 22.

Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany; Center for Biochemistry, Medical Faculty and University Hospital Cologne, University of Cologne, Cologne, Germany. Electronic address:

Energy metabolism and extracellular matrix (ECM) function together orchestrate and maintain tissue organization, but crosstalk between these processes is poorly understood. Here, we used single-cell RNA-Seq (scRNA-Seq) analysis to uncover the importance of the mitochondrial respiratory chain for ECM homeostasis in mature cartilage. This tissue produces large amounts of a specialized ECM to promote skeletal growth during development and maintain mobility throughout life. A combined approach of high-resolution scRNA-Seq, mass spectrometry/matrisome analysis, and atomic force microscopy was applied to mutant mice with cartilage-specific inactivation of respiratory chain function. This genetic inhibition in cartilage results in the expansion of a central area of 1-month-old mouse femur head cartilage, showing disorganized chondrocytes and increased deposition of ECM material. scRNA-Seq analysis identified a cell cluster-specific decrease in mitochondrial DNA-encoded respiratory chain genes and a unique regulation of ECM-related genes in nonarticular chondrocytes. These changes were associated with alterations in ECM composition, a shift in collagen/noncollagen protein content, and an increase of collagen crosslinking and ECM stiffness. These results demonstrate that mitochondrial respiratory chain dysfunction is a key factor that can promote ECM integrity and mechanostability in cartilage and presumably also in many other tissues.
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http://dx.doi.org/10.1016/j.jbc.2021.101224DOI Listing
September 2021

3D Printing of Antibacterial, Biocompatible, and Biomimetic Hybrid Aerogel-Based Scaffolds with Hierarchical Porosities via Integrating Antibacterial Peptide-Modified Silk Fibroin with Silica Nanostructure.

ACS Biomater Sci Eng 2021 09 20;7(9):4545-4556. Epub 2021 Aug 20.

Department of Chemistry, Institute of Inorganic Chemistry, University of Cologne, Greinstraße 6, 50939 Cologne, Germany.

Scaffold-mediated tissue engineering has become a golden solution for the regeneration of damaged bone tissues that lack self-regeneration capability. A successful scaffold in bone tissue engineering comprises a multitude of suitable biological, microarchitectural, and mechanical properties acting as different signaling cues for the cells to mediate the new tissue formation. Therefore, careful design of bioactive scaffold macro- and microstructures in multiple length scales and biophysical properties fulfilling the tissue repair demands are necessary yet challenging to achieve. Herein, we have developed an antibacterial and biocompatible silica-silk fibroin (SF) gel-based ink through novel yet simple chemical approaches of sol-gel and self-assembly followed by processing the obtained gels as three-dimensional (3D) hybrid aerogel-based scaffolds exploiting the advanced materials design approaches of micro-extrusion-based 3D printing, and directional freeze-casting/drying approaches. As the main constituent of the hybrid biocompatible scaffold of this study, we used the SF extracted from silkworm cocoon. However, to increase the cell responsivity and bactericidal efficiency of the final scaffold, thiol-ended antimicrobial and cell adhesive peptide sequence (SH-CM-RGD) was conjugated to silica-SF hybrid gels via covalent attachment using a spacer molecule through either preprint (prior to sol-gel) or during the post-printing steps on the previously printed silica-SF gel. In the next step, the hybrid Silica-SF-CM-RGD hydrogel ink was 3D-printed into the construct with interconnected porous structure with hierarchically organized porosity and a combination of several promising properties. Namely, due to the covalent linkage of the antibacterial peptide to the SF, the scaffold shows potent bactericidal efficiency toward Gram-positive and Gram-negative bacteria. Moreover, nanostructured silica components in the 3D-printed composites could intertwine with SF-CM-RGD to support the mechanical properties in the final scaffold and the final osteoconductivity of the scaffold. This study supports the promising properties of 3D-printed silica-SF-based hybrid aerogels constructs for repairing bone defect.
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http://dx.doi.org/10.1021/acsbiomaterials.1c00483DOI Listing
September 2021

Effects of long-term immobilisation on endomysium of the soleus muscle in humans.

Exp Physiol 2021 Oct 24;106(10):2038-2045. Epub 2021 Aug 24.

Institute of Aerospace Medicine, German Aerospace Center (DLR), Cologne, Germany.

New Findings: What is the central question of this study? While muscle fibre atrophy in response to immobilisation has been extensively examined, intramuscular connective tissue, particularly endomysium, has been largely neglected: does endomysium content of the soleus muscle increase during bed rest? What is the main finding and its importance? Absolute endomysium content did not change, and previous studies reporting an increase are explicable by muscle fibre atrophy. It must be expected that even a relative connective tissue accumulation will lead to an increase in muscle stiffness.

Abstract: Muscle fibres atrophy during conditions of disuse. Whilst animal data suggest an increase in endomysium content with disuse, that information is not available for humans. We hypothesised that endomysium content increases during immobilisation. To test this hypothesis, biopsy samples of the soleus muscle obtained from 21 volunteers who underwent 60 days of bed rest were analysed using immunofluorescence-labelled laminin γ-1 to delineate individual muscle fibres as well as the endomysium space. The endomysium-to-fibre-area ratio (EFAr, as a percentage) was assessed as a measure related to stiffness, and the endomysium-to-fibre-number ratio (EFNr) was calculated to determine whether any increase in EFAr was absolute, or could be attributed to muscle fibre shrinkage. As expected, we found muscle fibre atrophy (P = 0.0031) that amounted to shrinkage by 16.6% (SD 28.2%) on day 55 of bed rest. ENAr increased on day 55 of bed rest (P < 0.001). However, when analysing EFNr, no effect of bed rest was found (P = 0.62). These results demonstrate that an increase in EFAr is likely to be a direct effect of muscle fibre atrophy. Based on the assumption that the total number of muscle fibres remains unchanged during 55 days of bed rest, this implies that the absolute amount of connective tissue in the soleus muscle remained unchanged. The increased relative endomysium content, however, could be functionally related to an increase in muscle stiffness.
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http://dx.doi.org/10.1113/EP089734DOI Listing
October 2021

Pivotal Role of Tenascin-W (-N) in Postnatal Incisor Growth and Periodontal Ligament Remodeling.

Front Immunol 2020 22;11:608223. Epub 2021 Jan 22.

Faculty of Medicine and University Hospital Cologne, Institute for Dental Research and Oral Musculoskeletal Biology, University of Cologne, Cologne, Germany.

The continuously growing mouse incisor provides a fascinating model for studying stem cell regulation and organ renewal. In the incisor, epithelial and mesenchymal stem cells assure lifelong tooth growth. The epithelial stem cells reside in a niche known as the cervical loop. Mesenchymal stem cells are located in the nearby apical neurovascular bundle and in the neural plexus. So far, little is known about extracellular cues that are controlling incisor stem cell renewal and guidance. The extracellular matrix protein tenascin-W, also known as tenascin-N (TNN), is expressed in the mesenchyme of the pulp and of the periodontal ligament of the incisor, and is closely associated with collagen 3 fibers. Here, we report for the first time the phenotype of tenascin-W/TNN deficient mice, which in a C57BL/6N background exhibit a reduced body weight and lifespan. We found major defects in the alveolar bone and periodontal ligament of the growing rodent incisors, whereas molars were not affected. The alveolar bone around the incisor was replaced by a dense scar-like connective tissue, enriched with newly formed nerve fibers likely leading to periodontal pain, less food intake and reduced body weight. Using soft food to reduce mechanical load on the incisor partially rescued the phenotype. hybridization and Gli1 reporter mouse experiments revealed decreased hedgehog signaling in the incisor mesenchymal stem cell compartment, which coordinates the development of mesenchymal stem cell niche. These results indicate that TNN deficiency in mice affects periodontal remodeling and increases nerve fiber branching. Through periodontal pain the food intake is reduced and the incisor renewal and the neurovascular sonic hedgehog secretion rate are reduced. In conclusion, tenascin-W/TNN seems to have a primary function in rapid periodontal tissue remodeling and a secondary function in mechanosensation.
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http://dx.doi.org/10.3389/fimmu.2020.608223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7862723PMC
June 2021

miR-127-3p Is an Epigenetic Activator of Myofibroblast Senescence Situated within the MicroRNA-Enriched Dlk1-Dio3‒Imprinted Domain on Mouse Chromosome 12.

J Invest Dermatol 2021 04 3;141(4S):1076-1086.e3. Epub 2020 Dec 3.

Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty, University of Cologne, Cologne, Germany; Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany. Electronic address:

During wound healing, fibroblasts differentiate into nonproliferative contractile myofibroblasts, contribute to skin repair, and eventually undergo apoptosis or become senescent. MicroRNAs are post-transcriptional regulators of gene expression networks that control cell fate and survival and may also regulate senescence. In this study, we determined the regulated microRNAs in myofibroblasts isolated from wounds and analyzed their role in senescent myofibroblast formation. Transcriptome profiling showed that a 200 kilobase pair region of the Dlk1-Dio3‒imprinted domain on mouse chromosome 12 encodes for most of the upregulated microRNAs in the entire genome of mouse myofibroblasts. Among those, miR-127-3p induced a myofibroblast-like phenotype associated with a block in proliferation. Molecular analysis revealed that miR-127-3p induced a prolonged cell cycle arrest with unique molecular features of senescence, including the activation of the senescence-associated β-galactosidase, increase in p53 and p21 levels, inhibition of lamin B1, proliferation factors, and the production of senescence-associated inflammatory and extracellular matrix‒remodeling components. Hence, miR-127-3p emerges as an epigenetic activator regulating the transition from repair to remodeling during skin wound healing but may also induce age-related defects, pathological scarring, and fibrosis, all linked to myofibroblast senescence.
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http://dx.doi.org/10.1016/j.jid.2020.11.011DOI Listing
April 2021

Augmentation of and Suppression of Expression in the Pituitary Gland of Female Annexin A5 Null Mouse.

J Endocr Soc 2020 Sep 16;4(9):bvaa096. Epub 2020 Jul 16.

Laboratory of Veterinary Physiology, School of Veterinary Medicine, Kitasato University, Aomori, Japan.

GnRH enhances the expression of annexin A5 (ANXA5) in pituitary gonadotropes, and ANXA5 enhances gonadotropin secretion. However, the impact of ANXA5 regulation on the expression of pituitary hormone genes remains unclear. Here, using quantitative PCR, we demonstrated that ANXA5 deficiency in female mice reduced the expression of and in their pituitary glands. Transcriptome analysis confirmed a specific increase in mRNA expression in addition to lower levels of expression in ANXA5-deficient female pituitary glands. This gene was then found to be a GnRH-inducible immediate early gene, and its increased expression caused protein to accumulate in the nucleus after administration of a GnRH agonist in LβT2 cells, which are an in vitro pituitary gonadotrope model. The increase in ANXA5 protein levels in LβT2 cells clearly suppressed expression. siRNA-mediated inhibition of expression increased expression. The results revealed that GnRH stimulates and sequentially. NR4A3 suppression of may be necessary for later massive secretion of FSH by GnRH in gonadotropes, and would be negatively regulated by ANXA5 to increase FSH secretion.
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http://dx.doi.org/10.1210/jendso/bvaa096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7448937PMC
September 2020

Proteasomal degradation induced by DPP9-mediated processing competes with mitochondrial protein import.

EMBO J 2020 10 20;39(19):e103889. Epub 2020 Aug 20.

Institute of Biochemistry, Redox Biochemistry, University of Cologne, Cologne, Germany.

Plasticity of the proteome is critical to adapt to varying conditions. Control of mitochondrial protein import contributes to this plasticity. Here, we identified a pathway that regulates mitochondrial protein import by regulated N-terminal processing. We demonstrate that dipeptidyl peptidases 8/9 (DPP8/9) mediate the N-terminal processing of adenylate kinase 2 (AK2) en route to mitochondria. We show that AK2 is a substrate of the mitochondrial disulfide relay, thus lacking an N-terminal mitochondrial targeting sequence and undergoing comparatively slow import. DPP9-mediated processing of AK2 induces its rapid proteasomal degradation and prevents cytosolic accumulation of enzymatically active AK2. Besides AK2, we identify more than 100 mitochondrial proteins with putative DPP8/9 recognition sites and demonstrate that DPP8/9 influence the cellular levels of a number of these proteins. Collectively, we provide in this study a conceptual framework on how regulated cytosolic processing controls levels of mitochondrial proteins as well as their dual localization to mitochondria and other compartments.
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http://dx.doi.org/10.15252/embj.2019103889DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527813PMC
October 2020

Ablation of the miRNA Cluster 24 Has Profound Effects on Extracellular Matrix Protein Abundance in Cartilage.

Int J Mol Sci 2020 Jun 9;21(11). Epub 2020 Jun 9.

Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty, University of Cologne, 50931 Cologne, Germany.

MicroRNAs (miRNAs) regulate cartilage differentiation and contribute to the onset and progression of joint degeneration. These small RNA molecules may affect extracellular matrix organization (ECM) in cartilage, but for only a few miRNAs has this role been defined . Previously, we showed that cartilage-specific genetic ablation of the Mirc24 cluster in mice leads to impaired cartilage development due to increased RAF/MEK/ERK pathway activation. Here, we studied the expression of the cluster in cartilage by LacZ reporter gene assays and determined its role for extracellular matrix homeostasis by proteome and immunoblot analysis. The cluster is expressed in prehypertrophic/hypertrophic chondrocytes of the growth plate and we now show that the cluster is also highly expressed in articular cartilage. Cartilage-specific loss of the cluster leads to increased proteoglycan 4 and matrix metallopeptidase 13 levels and decreased aggrecan and collagen X levels in epiphyseal cartilage. Interestingly, these changes are linked to a decrease in SRY-related HMG box-containing (SOX) transcription factors 6 and 9, which regulate ECM production in chondrocytes. Our data suggests that the Mirc24 cluster is important for ECM homoeostasis and the expression of transcriptional regulators of matrix production in cartilage.
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http://dx.doi.org/10.3390/ijms21114112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7312048PMC
June 2020

Angiogenic Potential of Tissue Engineered Cartilage From Human Mesenchymal Stem Cells Is Modulated by Indian Hedgehog and Serpin E1.

Front Bioeng Biotechnol 2020 17;8:327. Epub 2020 Apr 17.

Department of Otorhinolaryngology, Head and Neck Surgery, Erasmus MC, University Medical Center, Rotterdam, Netherlands.

With rising demand for cartilage tissue repair and replacement, the differentiation of mesenchymal stem cells (BMSCs) into cartilage tissue forming cells provides a promising solution. Often, the BMSC-derived cartilage does not remain stable and continues maturing to bone through the process of endochondral ossification . Similar to the growth plate, invasion of blood vessels is an early hallmark of endochondral ossification and a necessary step for completion of ossification. This invasion originates from preexisting vessels that expand via angiogenesis, induced by secreted factors produced by the cartilage graft. In this study, we aimed to identify factors secreted by chondrogenically differentiated bone marrow-derived human BMSCs to modulate angiogenesis. The secretome of chondrogenic pellets at day 21 of the differentiation program was collected and tested for angiogenic capacity using endothelial migration and proliferation assays as well as the chick chorioallantoic membrane (CAM) assay. Taken together, these assays confirmed the pro-angiogenic potential of the secretome. Putative secreted angiogenic factors present in this medium were identified by comparative global transcriptome analysis between murine growth plate cartilage, human chondrogenic BMSC pellets and human neonatal articular cartilage. We then verified by PCR eight candidate angiogenesis modulating factors secreted by differentiated BMSCs. Among those, Serpin E1 and Indian Hedgehog (IHH) had a higher level of expression in BMSC-derived cartilage compared to articular chondrocyte derived cartilage. To understand the role of these factors in the pro-angiogenic secretome, we used neutralizing antibodies to functionally block them in the conditioned medium. Here, we observed a 1.4-fold increase of endothelial cell proliferation when blocking IHH and 1.5-fold by Serpin E1 blocking compared to unblocked control conditioned medium. Furthermore, endothelial migration was increased 1.9-fold by Serpin E1 blocking and 2.7-fold by IHH blocking. This suggests that the pro-angiogenic potential of chondrogenically differentiated BMSC secretome could be further augmented through inhibition of specific factors such as IHH and Serpin E1 identified as anti-angiogenic factors.
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http://dx.doi.org/10.3389/fbioe.2020.00327DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7180203PMC
April 2020

Altered molecular signatures during kidney development after intrauterine growth restriction of different origins.

J Mol Med (Berl) 2020 03 1;98(3):395-407. Epub 2020 Feb 1.

Department of Pediatrics and Adolescent Medicine, University of Cologne, Medical Faculty and University Hospital Cologne, Cologne, Germany.

This study was performed to identify transcriptional alterations in male intrauterine growth restricted (IUGR) rats during and at the end of nephrogenesis in order to generate hypotheses which molecular mechanisms contribute to adverse kidney programming. IUGR was induced by low protein (LP) diet throughout pregnancy, bilateral uterine vessel ligation (LIG), or intrauterine stress (IUS) by sham operation. Offspring of unimpaired dams served as controls. Significant acute kidney damage was ruled out by negative results for proteins indicative of ER-stress, autophagy, apoptosis, or infiltration with macrophages. Renal gene expression was examined by transcriptome microarrays, demonstrating 53 (LP, n = 12; LIG, n = 32; IUS, n = 9) and 134 (LP, n = 10; LIG, n = 41; IUS, n = 83) differentially expressed transcripts on postnatal days (PND) 1 and 7, respectively. Reduced Pilra (all IUGR groups, PND 7), Nupr1 (LP and LIG, PND 7), and Kap (LIG, PND 1) as well as increased Ccl20, S100a8/a9 (LIG, PND 1), Ifna4, and Ltb4r2 (IUS, PND 7) indicated that inflammation-related molecular dysregulation could be a "common" feature after IUGR of different origins. Network analyses of transcripts and predicted upstream regulators hinted at proinflammatory adaptions mainly in LIG (arachidonic acid-binding, neutrophil aggregation, toll-like-receptor, NF-kappa B, and TNF signaling) and dysregulation of AMPK and PPAR signaling in LP pups. The latter may increase susceptibility towards obesity-associated kidney damage. Western blots of the most prominent predicted upstream regulators confirmed significant dysregulation of RICTOR in LP (PND 7) and LIG pups (PND 1), suggesting that mTOR-related processes could further modulate kidney programming in these groups of IUGR pups. KEY MESSAGES: Inflammation-related transcripts are dysregulated in neonatal IUGR rat kidneys. Upstream analyses indicate renal metabolic dysregulation after low protein diet. RICTOR is dysregulated after low protein diet and uterine vessel ligation.
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http://dx.doi.org/10.1007/s00109-020-01875-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7080693PMC
March 2020

Gene Expression Profiling of the Extracellular Matrix Signature in Macrophages of Different Activation Status: Relevance for Skin Wound Healing.

Int J Mol Sci 2019 Oct 14;20(20). Epub 2019 Oct 14.

Center for Biochemistry, Medical Faculty, University of Cologne, 50931 Cologne, Germany.

The extracellular matrix (ECM) provides structural support for tissue architecture and is a major effector of cell behavior during skin repair and inflammation. Macrophages are involved in all stages of skin repair but only limited knowledge exists about macrophage-specific expression and regulation of ECM components. In this study, we used transcriptome profiling and bioinformatic analysis to define the unique expression of ECM-associated genes in cultured macrophages. Characterization of the matrisome revealed that most genes were constitutively expressed and that several genes were uniquely regulated upon interferon gamma (IFNγ) and dexamethasone stimulation. Among those core matrisome and matrisome-associated components transforming growth factor beta (TGFβ)-induced, matrix metalloproteinase 9 (MMP9), elastin microfibril interfacer (EMILIN)-1, netrin-1 and gliomedin were also present within the wound bed at time points that are characterized by profound macrophage infiltration. Hence, macrophages are a source of ECM components in vitro as well as during skin wound healing, and identification of these matrisome components is a first step to understand the role and therapeutic value of ECM components in macrophages and during wound healing.
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http://dx.doi.org/10.3390/ijms20205086DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6829210PMC
October 2019

Respiratory chain inactivation links cartilage-mediated growth retardation to mitochondrial diseases.

J Cell Biol 2019 06 13;218(6):1853-1870. Epub 2019 May 13.

Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Faculty of Medicine, University of Cologne, Cologne, Germany

In childhood, skeletal growth is driven by transient expansion of cartilage in the growth plate. The common belief is that energy production in this hypoxic tissue mainly relies on anaerobic glycolysis and not on mitochondrial respiratory chain (RC) activity. However, children with mitochondrial diseases causing RC dysfunction often present with short stature, which indicates that RC activity may be essential for cartilage-mediated skeletal growth. To elucidate the role of the mitochondrial RC in cartilage growth and pathology, we generated mice with impaired RC function in cartilage. These mice develop normally until birth, but their later growth is retarded. A detailed molecular analysis revealed that metabolic signaling and extracellular matrix formation is disturbed and induces cell death at the cartilage-bone junction to cause a chondrodysplasia-like phenotype. Hence, the results demonstrate the overall importance of the metabolic switch from fetal glycolysis to postnatal RC activation in growth plate cartilage and explain why RC dysfunction can cause short stature in children with mitochondrial diseases.
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http://dx.doi.org/10.1083/jcb.201809056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6548139PMC
June 2019

Novel DRAQ5™/SYTOX® Blue Based Flow Cytometric Strategy to Identify and Characterize Stem Cells in Human Breast Milk.

Cytometry B Clin Cytom 2019 11 26;96(6):480-489. Epub 2018 Nov 26.

Medical Faculty, Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, University of Cologne, Cologne, Germany.

Background: Human breast milk could be an important stem cell source for the development of newborn and preterm infants, but quantitative data on the stem cell content in breast milk at various gestational stages are needed to determine the clinical value of breast milk as a source of stem cells. Breast milk also contains milk fat globules, lipid droplets of different sizes, debris and dead cells and these components hamper flow cytometry analysis of human breast milk samples.

Methods: Here, we originally used standard protocols for flow cytometry to characterize cell populations in human breast milk but failed to discriminate between cells and noncellular components. We then applied a centrifugation protocol to separate cream and skim milk from the cell-containing pellet and used a novel staining protocol with DRAQ5™ and SYTOX® blue dye as well as antibodies to characterize cells within the pellet fraction.

Results: Flow cytometry analysis identified viable DRAQ5™ /SYTOX® Blue cells and determined the content of CD11b monocytes and TRA-1-81 putative stem cells in human breast milk samples.

Conclusions: Hence, we developed a novel and reliable flow cytometry based-approach to quantify subpopulation of cells in human breast milk with a high content of milk fat globules, lipid droplets, and particles. This approach will improve the identification and quantification of breast milk cells and allow standardizing the flow cytometry-based evaluation of the stem cell content. © 2018 International Clinical Cytometry Society.
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http://dx.doi.org/10.1002/cyto.b.21748DOI Listing
November 2019

Detection of annexin A8 antibodies in serum of patients with antiphospholipid syndrome.

Biochem Med (Zagreb) 2018 Oct;28(3):030703

Institute for Clinical Chemistry, Medical Faculty, University of Cologne, Cologne, Germany.

Introduction: Antibodies specific for annexin A8 (AnxA8) have not been investigated in patients suffering from antiphospholipid syndrome (APS) yet. The aim of this study was to compare the presence of AnxA8 antibodies in serum of APS patients with that of age-matched healthy controls and to investigate whether AnxA8 antibodies are potential biomarkers for APS.

Materials And Methods: We enrolled 22 APS patients and 22 healthy controls in this case-control study. We used sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoblot to investigate the presence of AnxA8 antibodies, and we applied enzyme-linked immunosorbent assay to investigate the presence of cardiolipin (CL) and beta-2-glycoprotein I (ß2GPI) antibodies.

Results: The serum of 9/22 APS patients showed AnxA8 IgG isotype antibody reactivity compared to serum of 2/22 healthy controls (P = 0.034). When we also included weak immunoblot signals, 12/22 APS patients exhibited AnxA8 IgG isotype antibody reactivity compared to 3/22 healthy controls (P = 0.005). We also investigated the presence of AnxA8 IgM isotype antibodies in the serum of APS patients but found no statistically significant difference between the APS patient group and healthy control group (P = 0.500). We further investigated the presence of ß2GPI and CL IgG and IgM isotype antibodies. AnxA8 IgG isotype antibodies were present in APS patients in a similar frequency as the APS "criteria" antibody against CL (P = 0.764).

Conclusion: We demonstrated that AnxA8 IgG isotype antibodies are potential biomarkers for the diagnosis of APS.
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http://dx.doi.org/10.11613/BM.2018.030703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6214702PMC
October 2018

Dual role of laminin‑511 in regulating melanocyte migration and differentiation.

Matrix Biol 2019 07 29;80:59-71. Epub 2018 Sep 29.

Translational Matrix Biology, Medical Faculty, University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany. Electronic address:

Laminins are the major basement membrane (BM) components and are heterotrimers composed of an α, a β and a γ chain. In skin, laminins are present in basement membranes surrounding vascular structures, nerves, adipose tissue and in the specialized junctional BM between the epidermis and dermis. The main laminin isoforms in the dermo-epidermal BM are laminin‑332, laminin‑511 and laminin‑211, the latter being restricted to hair follicles (HFs). The laminin γ1 chain is the most abundant γ chain; its global ablation in mice leads to early embryonic lethality at E5.5. To elucidate the cellular function of the γ1 chain in skin, we generated mice with keratinocyte-specific deletion of this chain (Lamc1) by using the keratin (K)14-Cre/loxP system. These mice showed delayed coat pigmentation despite normal melanocyte counts in the skin. However, levels of differentiation-specific melanocyte enzymes TRP‑1, TRP‑2 and tyrosinase were reduced in Lamc1 mice, and melanocytes failed to migrate to their differentiation niche in HFs and accumulated in the IFE. These results suggested that the pigmentation defect results from impaired melanocyte migration. The impaired migratory capacity of melanocytes is due to the altered composition of laminins in the BM of Lamc1 mice: Loss of keratinocyte-derived pro-migratory laminin‑511 is not compensated by ectopically deposited fibroblast-derived laminin‑211. Furthermore, contact of melanocytes with recombinant laminin‑511, but not with laminin‑211, induces the expression of the chemokine receptor CXCR4 on melanocytes, needed for SDF‑1 (stromal cell‑derived factor‑1)-mediated migration into HFs. We here demonstrate that laminin‑511 controls the differentiation of melanocytes by regulating their migration from the epidermis into HFs and by activating CXCR4 expression on melanocytes required for their recruitment into HFs in an SDF‑1-dependent manner.
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http://dx.doi.org/10.1016/j.matbio.2018.09.006DOI Listing
July 2019

Depletion of Collagen IX Alpha1 Impairs Myeloid Cell Function.

Stem Cells 2018 11 11;36(11):1752-1763. Epub 2018 Sep 11.

Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty, University of Cologne, Cologne, Germany.

The trabecular extracellular matrix (ECM) forms a three-dimensional scaffold to stabilize the bone marrow, provide substrates for cell-matrix interactions and retain, present or release signals to modulate hematopoietic stem and progenitor cell development. However, the impact of trabecular ECM components on hematopoiesis has been poorly studied. Using collagen IX alpha1 - deficient (Col9a1 ) mice, we revealed that a lack of collagen IX alpha1 results in a disorganized trabecular network enriched in fibronectin, and in a reduction in myeloid cells, which was accompanied by a decrease in colony-stimulating factor 1 receptor expression on monocytes from the bone marrow. In contrast, B-cell numbers in the bone marrow and T-cell numbers in the thymus remained unchanged. Alterations in the bone marrow microenvironment may not only reduce myeloid cell numbers, but also have long-term implications for myeloid cell function. Mice were infected with Listeria moncytogenes to analyze the function of myeloid cells. In this case, an inadequate macrophage-dependent clearance of bacterial infections was observed in Col9a1 mice in vivo. This was mainly caused by an impaired interferon-gamma/tumor necrosis factor-alpha-mediated activation of macrophages. The loss of collagen IX alpha1 therefore destabilizes the trabecular bone network, impairs myeloid cell differentiation, and affects the innate immune response against Listeria. Stem Cells 2018;36:1752-1763.
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http://dx.doi.org/10.1002/stem.2892DOI Listing
November 2018

Identification of TGFβ-related genes regulated in murine osteoarthritis and chondrocyte hypertrophy by comparison of multiple microarray datasets.

Bone 2018 11 21;116:67-77. Epub 2018 Jul 21.

Department of Orthopedics, Erasmus MC University Medical Center, Rotterdam, the Netherlands; Department of Otorhinolaryngology, Erasmus MC University Medical Center, Rotterdam, the Netherlands. Electronic address:

Objective: Osteoarthritis (OA) is a joint disease characterized by progressive degeneration of articular cartilage. Some features of OA, including chondrocyte hypertrophy and focal calcification of articular cartilage, resemble the endochondral ossification processes. Alterations in transforming growth factor β (TGFβ) signaling have been associated with OA as well as with chondrocyte hypertrophy. Our aim was to identify novel candidate genes implicated in chondrocyte hypertrophy during OA pathogenesis by determining which TGFβ-related genes are regulated during murine OA and endochondral ossification.

Methods: A list of 580 TGFβ-related genes, including TGFβ signaling pathway components and TGFβ-target genes, was generated. Regulation of these TGFβ-related genes was assessed in a microarray of murine OA cartilage: 1, 2 and 6 weeks after destabilization of the medial meniscus (DMM). Subsequently, genes regulated in the DMM model were studied in two independent murine microarray datasets on endochondral ossification: the growth plate and transient embryonic cartilage (joint development).

Results: A total of 106 TGFβ-related genes were differentially expressed in articular cartilage of DMM-operated mice compared to sham-control. From these genes, 43 were similarly regulated during chondrocyte hypertrophy in the growth plate or embryonic joint development. Among these 43 genes, 18 genes have already been associated with OA. The remaining 25 genes were considered as novel candidate genes involved in OA pathogenesis and endochondral ossification. In supplementary data of published human OA microarrays we found indications that 15 of the 25 novel genes are indeed regulated in articular cartilage of human OA patients.

Conclusion: By focusing on TGFβ-related genes during OA and chondrocyte hypertrophy in mice, we identified 18 known and 25 new candidate genes potentially implicated in phenotypical changes in chondrocytes leading to OA. We propose that 15 of these candidates warrant further investigation as therapeutic target for OA as they are also regulated in articular cartilage of OA patients.
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http://dx.doi.org/10.1016/j.bone.2018.07.008DOI Listing
November 2018

Annexin A5 Involvement in Bone Overgrowth at the Enthesis.

J Bone Miner Res 2018 08;33(8):1532-1543

Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama, Japan.

Little is known about the molecular mechanisms of enthesis formation in mature animals. Here, we report that annexin A5 (Anxa5) plays a critical role in the regulation of bone ridge outgrowth at the entheses. We found that Anxa5 is highly expressed in the entheses of postnatal and adult mice. In Anxa5-deficient (Anxa5 ) mice, the sizes of bone ridge outgrowths at the entheses of the tibias and femur were increased after age 7 weeks. Bone overgrowth was not observed at the fibrous enthesis where the fibrocartilage layer does not exist. More ALP-expressing cells were observed in the fibrocartilage layer in Anxa5 mice than in wild-type (WT) mice. Calcein and Alizarin Red double labeling revealed more mineralized areas in Anxa5 mice than WT mice. To examine the effects of mechanical forces, we performed tenotomy in which transmission of contractile forces by the tibial muscle was impaired by surgical muscle release. In tenotomized mice, bone overgrowth at the enthesis in Anxa5 mice was decreased to a level comparable to that in WT mice at 8 weeks after the operation. The tail-suspended mice also showed a decrease in bone overgrowth to similar levels in Anxa5 and WT mice at 8 weeks after hindlimb unloading. These results suggest that bone overgrowth at the enthesis requires mechanical forces. We further examined effects of Anxa5 gene knockdown (KD) in primary cultures of osteoblasts, chondrocytes, and tenocytes in vitro. Anxa5 KD increased ALP expression in tenocytes and chondrocytes but not in osteoblasts, suggesting that increased ALP activity in the fibrocartilaginous tissue in Anxa5 mice is directly caused by Anxa5 deletion in tenocytes or fibrocartilage cells. These data indicate that Anxa5 prevents bone overgrowth at the enthesis, whose formation is mediated through mechanical forces and modulating expression of mineralization regulators. © 2018 American Society for Bone and Mineral Research.
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http://dx.doi.org/10.1002/jbmr.3453DOI Listing
August 2018

Post-weaning epiphysiolysis causes distal femur dysplasia and foreshortened hindlimbs in fetuin-A-deficient mice.

PLoS One 2017 31;12(10):e0187030. Epub 2017 Oct 31.

Biointerface Laboratory, Helmholtz Institute for Biomedical Engineering, RWTH Aachen University Hospital, Aachen, Germany.

Fetuin-A / α2-Heremans-Schmid-glycoprotein (gene name Ahsg) is a systemic inhibitor of ectopic calcification. Due to its high affinity for calcium phosphate, fetuin-A is highly abundant in mineralized bone matrix. Foreshortened femora in fetuin-A-deficient Ahsg-/- mice indicated a role for fetuin-A in bone formation. We studied early postnatal bone development in fetuin-A-deficient mice and discovered that femora from Ahsg-/- mice exhibited severely displaced distal epiphyses and deformed growth plates, similar to the human disease slipped capital femoral epiphysis (SCFE). The growth plate slippage occurred in 70% of Ahsg-/- mice of both sexes around three weeks postnatal. At this time point, mice weaned and rapidly gained weight and mobility. Epiphysis slippage never occurred in wildtype and heterozygous Ahsg+/- mice. Homozygous fetuin-A-deficient Ahsg-/- mice and, to a lesser degree, heterozygous Ahsg+/- mice showed lesions separating the proliferative zone from the hypertrophic zone of the growth plate. The hypertrophic growth plate cartilage in long bones from Ahsg-/- mice was significantly elongated and V-shaped until three weeks of age and thus prior to the slippage. Genome-wide transcriptome analysis of laser-dissected distal femoral growth plates from 13-day-old Ahsg-/- mice revealed a JAK-STAT-mediated inflammatory response including a 550-fold induction of the chemokine Cxcl9. At this stage, vascularization of the elongated growth plates was impaired, which was visualized by immunofluorescence staining. Thus, fetuin-A-deficient mice may serve as a rodent model of growth plate pathologies including SCFE and inflammatory cartilage degradation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0187030PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5663435PMC
November 2017

Imbalance of Mitochondrial Respiratory Chain Complexes in the Epidermis Induces Severe Skin Inflammation.

J Invest Dermatol 2018 01 1;138(1):132-140. Epub 2017 Sep 1.

Center for Physiology and Pathophysiology, Institute of Vegetative Physiology, University of Köln, Köln, Germany.

Accumulation of large-scale mitochondrial DNA (mtDNA) deletions and chronic, subclinical inflammation are concomitant during skin aging, thus raising the question of a causal link. To approach this, we generated mice expressing a mutant mitochondrial helicase (K320E-TWINKLE) in the epidermis to accelerate the accumulation of mtDNA deletions in this skin compartment. Mice displayed low amounts of large-scale deletions and a dramatic depletion of mtDNA in the epidermis and showed macroscopic signs of severe skin inflammation. The mtDNA alterations led to an imbalanced stoichiometry of mitochondrial respiratory chain complexes, inducing a unique combination of cytokine expression, causing a severe inflammatory phenotype, with massive immune cell infiltrates already before birth. Altogether, these data unraveled a previously unknown link between an imbalanced stoichiometry of the mitochondrial respiratory chain complexes and skin inflammation and suggest that severe respiratory chain dysfunction, as observed in few cells leading to a mosaic in aged tissues, might be involved in the development of chronic subclinical inflammation.
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http://dx.doi.org/10.1016/j.jid.2017.08.019DOI Listing
January 2018

miR-322 stabilizes MEK1 expression to inhibit RAF/MEK/ERK pathway activation in cartilage.

Development 2017 10 29;144(19):3562-3577. Epub 2017 Aug 29.

Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty, University of Cologne, Cologne 50931, Germany

Cartilage originates from mesenchymal cell condensations that differentiate into chondrocytes of transient growth plate cartilage or permanent cartilage of the articular joint surface and trachea. MicroRNAs fine-tune the activation of entire signaling networks and thereby modulate complex cellular responses, but so far only limited data are available on miRNAs that regulate cartilage development. Here, we characterize a miRNA that promotes the biosynthesis of a key component in the RAF/MEK/ERK pathway in cartilage. Specifically, by transcriptome profiling we identified miR-322 to be upregulated during chondrocyte differentiation. Among the various miR-322 target genes in the RAF/MEK/ERK pathway, only was identified as a regulated target in chondrocytes. Surprisingly, an increased concentration of miR-322 stabilizes mRNA to raise protein levels and dampen ERK1/2 phosphorylation, while cartilage-specific inactivation of miR322 in mice linked the loss of miR-322 to decreased MEK1 levels and to increased RAF/MEK/ERK pathway activation. Such mice died perinatally due to tracheal growth restriction and respiratory failure. Hence, a single miRNA can stimulate the production of an inhibitory component of a central signaling pathway to impair cartilage development.
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http://dx.doi.org/10.1242/dev.148429DOI Listing
October 2017

The LIM-Only Protein Four and a Half LIM Domain Protein 2 Attenuates Development of Psoriatic Arthritis by Blocking Adam17-Mediated Tumor Necrosis Factor Release.

Am J Pathol 2017 Nov 18;187(11):2388-2398. Epub 2017 Aug 18.

Institute of Virology, Westfaelische Wilhelms University, Muenster, Germany. Electronic address:

Four and a half LIM domain protein 2 (Fhl2) is an intracellular adaptor molecule with a high protein-protein interaction capacity. It acts as a modulator of several signaling molecules in the cytosol and as a cofactor of transcription in the nucleus. Recent studies suggest the role of Fhl2 in tissue repair and the anti-inflammatory response. Herein, we show that Fhl2-deficient mice develop a more severe psoriatic arthritis disease under induction of the inducible human tumor necrosis factor (hTNF) transgene than wild-type mice. The disease was accompanied by increased infiltration of activated macrophages and T regulatory cells in skin and digit joints as well as by increased expression of matrix metalloproteases and bone-specific proteases. The more severe pathogenesis of psoriatic arthritis in Fhl2 knockout mice coincided with enhanced levels of soluble hTNF cytokine, but surprisingly not with transcription of the hTNF transgene. Studying the shedding of cell membrane-bound hTNF by Adam17, a known Fhl2 interacting protein, revealed an enhanced release of TNF in the absence of Fhl2. In summary, our results show that Fhl2 anticipates the emerging inflammation and specifically the development of psoriatic arthritis by impeding the Adam17-mediated release of TNF.
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http://dx.doi.org/10.1016/j.ajpath.2017.07.015DOI Listing
November 2017

Identification of a myofibroblast-specific expression signature in skin wounds.

Matrix Biol 2018 01 7;65:59-74. Epub 2017 Aug 7.

Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty, University of Cologne, Cologne, Germany; Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany. Electronic address:

After skin injury fibroblasts migrate into the wound and transform into contractile, extracellular matrix-producing myofibroblasts to promote skin repair. Persistent activation of myofibroblasts can cause excessive fibrotic reactions, but the underlying mechanisms are not fully understood. We used SMA-GFP transgenic mice to study myofibroblast recruitment and activation in skin wounds. Myofibroblasts were initially recruited to wounds three days post injury, their number reached a maximum after seven days and subsequently declined. Expression profiling showed that 1749 genes were differentially expressed in sorted myofibroblasts from wounds seven days post injury. Most of these genes were linked with the extracellular region and cell periphery including genes encoding for extracellular matrix proteins. A unique panel of core matrisome and matrisome-associated genes was differentially expressed in myofibroblasts and several genes not yet known to be linked to myofibroblast-mediated wound healing were found (e.g. Col24a1, Podnl1, Bvcan, Tinagl1, Thbs3, Adamts16, Adamts19, Cxcl's, Ccl's). In addition, a complex network of G protein-coupled signaling events was regulated in myofibroblasts (e.g. Adcy1, Plbc4, Gnas). Hence, this first characterization of a myofibroblast-specific expression profile at the peak of in situ granulation tissue formation provides important insights into novel target genes that may control excessive ECM deposition during fibrotic reactions.
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http://dx.doi.org/10.1016/j.matbio.2017.07.005DOI Listing
January 2018

Mimicking Angiogenesis : Three-dimensional Co-culture of Vascular Endothelial Cells and Perivascular Cells in Collagen Type I Gels.

Bio Protoc 2017 Apr 20;7(8):e2247. Epub 2017 Apr 20.

Medical Faculty, Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Cologne, Germany.

Angiogenesis defines the process of formation of new vascular structures form existing blood vessels, involved during development, repair processes like wound healing but also linked to pathological changes. During angiogenic processes, endothelial cells build a vascular network and recruit perivascular cells to form mature, stable vessels. Endothelial cells and perivascular cells secret and assemble a vascular basement membrane and interact via close cell-cell contacts. To mimic these processes we have developed a versatile three-dimensional culture system where perivascular cells (PVC) are co-cultured with human umbilical cord vascular endothelial cells (HUVEC) in a collagen type I gel. This co-culture system can be used to determine biochemical and cellular processes during neoangiogenic events with a wide range of analyses options.
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http://dx.doi.org/10.21769/BioProtoc.2247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8410413PMC
April 2017

Macrophage-mediated psoriasis can be suppressed by regulatory T lymphocytes.

J Pathol 2016 11;240(3):366-377

Institute of Molecular Virology, ZMBE, University Hospital Muenster, 48149, Muenster, Germany.

We recently described an inducible human TNF transgenic mouse line (ihTNFtg) that develops psoriasis-like arthritis after doxycycline stimulation and analysed the pathogenesis of arthritis in detail. Here, we show that the skin phenotype of these mice is characterized by hyperproliferation and aberrant activation of keratinocytes, induction of pro-inflammatory cytokines, and infiltration with Th1 and Treg lymphocytes, particularly with macrophage infiltration into lesional skin, thus pointing to a psoriasis-like phenotype. To reveal the contribution of T cells and macrophages to the development of TNF-mediated psoriasis, ihTNFtg mice were crossbred into RAG1 mice lacking mature T and B cells. Surprisingly, the psoriatic phenotype in the double mutants was not reduced; rather, it was enhanced. The skin showed significantly increased inflammation and in particular, increased infiltration by macrophages. Consequently, depletion of macrophages in RAG1 or wild-type mice led to decreased disease severity. On the contrary, depletion of Treg cells in wild-type mice increased both psoriasis and the number of infiltrating macrophages, while adoptive transfer of Foxp3-positive cells into RAG1 or wild-type mice decreased both the development of psoriasis and macrophage infiltration. Thus, we conclude that Treg lymphocytes inhibit the pro-inflammatory activity of macrophages, which are the major immune effector cells in hTNF-mediated psoriasis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/path.4786DOI Listing
November 2016

Identification of a reference gene for the quantification of mRNA and miRNA expression during skin wound healing.

Connect Tissue Res 2017 Mar 7;58(2):196-207. Epub 2016 Jul 7.

a Department of Pediatrics and Adolescent Medicine, Experimental Neonatology, Medical Faculty , University of Cologne , Cologne , Germany.

Aim: Wound healing is a coordinated process to restore tissue homeostasis and reestablish the protective barrier of the skin. miRNAs may modulate the expression of target genes to contribute to repair processes, but due to the complexity of the tissue it is challenging to quantify gene expression during the distinct phases of wound repair. Here, we aimed to identify a common reference gene to quantify changes in miRNA and mRNA expression during skin wound healing.

Methods: Quantitative real-time PCR and bioinformatic analysis tools were used to identify suitable reference genes during skin repair and their reliability was tested by studying the expression of mRNAs and miRNAs.

Results: Morphological assessment of wounds showed that the injury model recapitulates the distinct phases of skin repair. Non-degraded RNA could be isolated from skin and wounds and used to study the expression of non-coding small nuclear RNAs during wound healing. Among those, RNU6B was most constantly expressed during skin repair. Using this reference gene we could confirm the transient upregulation of IL-1β and PTPRC/CD45 during the early phase as well as the increased expression of collagen type I at later stages of repair and validate the differential expression of miR-204, miR-205, and miR-31 in skin wounds. In contrast to Gapdh the normalization to multiple reference genes gave a similar outcome.

Conclusion: RNU6B is an accurate alternative normalizer to quantify mRNA and miRNA expression during the distinct phases of skin wound healing when analysis of multiple reference genes is not feasible.
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http://dx.doi.org/10.1080/03008207.2016.1210606DOI Listing
March 2017

Detection of multiple annexin autoantibodies in a patient with recurrent miscarriages, fulminant stroke and seronegative antiphospholipid syndrome.

Biochem Med (Zagreb) 2016 ;26(2):272-8

Institute for Clinical Chemistry, University of Cologne, Germany.

Anti-phospholipid syndrome (APS) is one of the main causes for recurrent miscarriages. The diagnosis of APS is based on the occurrence of clinical symptoms such as thrombotic events or obstetric complications as well as the detection of antiphospholipid antibodies directed against β2-glycoprotein I and cardiolipin, or a positive lupus anticoagulant assay. However, there is a subpopulation of patients with clinical symptoms of APS, but the lack of serological markers (seronegative APS). In addition, a large proportion of patients with unexplained recurrent miscarriages exist. These cases may be attributed, at least in part, to a seronegative APS.
The presence of autoantibodies against annexins is potentially associated with APS. Here we used immunoassays and immunoblots to detect autoantibodies directed against annexin A1-5, and A8, respectively, in a patient with a seronegative APS and a history of six recurrent pregnancy losses and fulminant stroke. We found strong IgM isotype antibody reactivity directed against annexin A2 and annexin A8, and moderate to weak IgM isotype antibody reactivity directed against annexin A1, A3, and A5. Further studies will evaluate the diagnostic value of IgM isotype antibodies against annexin A1-A5, and A8 for seronegative APS and recurrent miscarriages.
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http://dx.doi.org/10.11613/BM.2016.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4910275PMC
August 2016

Imatinib Triggers Phagolysosome Acidification and Antimicrobial Activity against Mycobacterium bovis Bacille Calmette-Guérin in Glucocorticoid-Treated Human Macrophages.

J Immunol 2016 07 27;197(1):222-32. Epub 2016 May 27.

Department of Dermatology, University of Cologne, Cologne 50937, Germany; Center for Molecular Medicine, University of Cologne, Cologne 50937, Germany;

Glucocorticoids are extensively used to treat inflammatory diseases; however, their chronic intake increases the risk for mycobacterial infections. Meanwhile, the effects of glucocorticoids on innate host responses are incompletely understood. In this study, we investigated the direct effects of glucocorticoids on antimycobacterial host defense in primary human macrophages. We found that glucocorticoids triggered the expression of cathelicidin, an antimicrobial critical for antimycobacterial responses, independent of the intracellular vitamin D metabolism. Despite upregulating cathelicidin, glucocorticoids failed to promote macrophage antimycobacterial activity. Gene expression profiles of human macrophages treated with glucocorticoids and/or IFN-γ, which promotes induction of cathelicidin, as well as antimycobacterial activity, were investigated. Using weighted gene coexpression network analysis, we identified a module of highly connected genes that was strongly inversely correlated with glucocorticoid treatment and associated with IFN-γ stimulation. This module was linked to the biological functions autophagy, phagosome maturation, and lytic vacuole/lysosome, and contained the vacuolar H(+)-ATPase subunit a3, alias TCIRG1, a known antimycobacterial host defense gene, as a top hub gene. We next found that glucocorticoids, in contrast with IFN-γ, failed to trigger expression and phagolysosome recruitment of TCIRG1, as well as to promote lysosome acidification. Finally, we demonstrated that the tyrosine kinase inhibitor imatinib induces lysosome acidification and antimicrobial activity in glucocorticoid-treated macrophages without reversing the anti-inflammatory effects of glucocorticoids. Taken together, we provide evidence that the induction of cathelicidin by glucocorticoids is not sufficient for macrophage antimicrobial activity, and identify the vacuolar H(+)-ATPase as a potential target for host-directed therapy in the context of glucocorticoid therapy.
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http://dx.doi.org/10.4049/jimmunol.1502407DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5536119PMC
July 2016

Skin Wound Repair Is Not Altered in the Absence of Endogenous AnxA1 or AnxA5, but Pharmacological Concentrations of AnxA4 and AnxA5 Inhibit Wound Hemostasis.

Cells Tissues Organs 2016 30;201(4):287-98. Epub 2016 Apr 30.

Skin injury induces the cell surface exposure of phosphatidylserine (PS) on damaged and dying cells to activate coagulation and repair processes. Annexins can bind to PS and may modulate the healing response. Here, we determine the relevance of annexins for skin wound healing using AnxA1- and AnxA5-deficient mice and recombinant annexins with distinct PS binding properties. Wound inflammation, closure and the formation of granulation tissue were not altered in AnxA1- or AnxA5-deficient mice or after increasing AnxA5 serum concentrations (100 nM) in wild-type mice. Increased serum concentrations (1 µM) of AnxA5 induced massive bleeding, but wound hemostasis was not delayed by AnxA1. Both annexins interact with PS, but only AnxA5 can form 2-dimensional (2D) arrays on the cell surface. The injection of an AnxA5 mutant that binds to PS but lacks the ability of 2D array formation failed to induce bleeding. 2D lattice-forming AnxA4, with high affinity to PS also caused bleeding, while hemostasis was not affected by AnxA8 with low affinity or the AnxA8 mutant with medium affinity for PS and the lack of 2D formation. Increased concentrations of AnxA4 and AnxA5 also delayed coagulation pathway activation in vitro. This effect was attenuated for the AnxA5 mutant as well as for AnxA1 and AnxA8. In conclusion, endogenous AnxA1 and AnxA5 are dispensable for wound hemostasis and repair, but pharmacologically excessive concentrations of AnxA4 and AnxA5 inhibit hemostasis in skin wounds.
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http://dx.doi.org/10.1159/000445106DOI Listing
March 2017

Erratum to: Induction of initial steps of angiogenic differentiation and maturation of endothelial cells by pericytes in vitro and the role of collagen IV.

Histochem Cell Biol 2016 May;145(5):527-9

Biomedical Research Centre, School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich, NR4 7TJ, UK.

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http://dx.doi.org/10.1007/s00418-016-1429-4DOI Listing
May 2016
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