Publications by authors named "Jürgen Brinckmann"

37 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jbc.2021.101224DOI Listing
September 2021

Fibroblast MMP14-Dependent Collagen Processing Is Necessary for Melanoma Growth.

Cancers (Basel) 2021 Apr 20;13(8). Epub 2021 Apr 20.

Department of Dermatology and Venereology, Faculty of Medicine, University of Cologne, Hospital Cologne, 50937 Cologne, Germany.

Skin homeostasis results from balanced synthesis and degradation of the extracellular matrix in the dermis. Deletion of the proteolytic enzyme MMP14 in dermal fibroblasts (MMP14) leads to a fibrotic skin phenotype with the accumulation of collagen type I, resulting from impaired proteolysis. Here, we show that melanoma growth in these mouse fibrotic dermal samples was decreased, paralleled by reduced tumor cell proliferation and vessel density. Using atomic force microscopy, we found increased peritumoral matrix stiffness of early but not late melanomas in the absence of fibroblast-derived MMP14. However, total collagen levels were increased at late melanoma stages in MMP14 mice compared to controls. In ex vivo invasion assays, melanoma cells formed smaller tumor islands in MMP14 skin, indicating that MMP14-dependent matrix accumulation regulates tumor growth. In line with these data, in vitro melanoma cell growth was inhibited in high collagen 3D spheroids or stiff substrates. Most importantly, in vivo induction of fibrosis using bleomycin reduced melanoma tumor growth. In summary, we show that MMP14 expression in stromal fibroblasts regulates melanoma tumor progression by modifying the peritumoral matrix and point to collagen accumulation as a negative regulator of melanoma.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers13081984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074311PMC
April 2021

Increased volume and collagen crosslinks drive soft tissue contribution to post-traumatic elbow contracture in an animal model.

J Orthop Res 2021 Aug 26;39(8):1800-1810. Epub 2020 Jun 26.

Department of Biomedical Engineering, Washington University in St. Louis, St. Louis, Missouri.

Post-traumatic joint contracture (PTJC) in the elbow is a biological problem with functional consequences. Restoring elbow motion after injury is a complex challenge because contracture is a multi-tissue pathology. We previously developed an animal model of elbow PTJC using Long-Evans rats and showed that the capsule and ligaments/cartilage were the primary soft tissues that caused persistent joint motion loss. The objective of this study was to evaluate tissue-specific changes within the anterior capsule and lateral collateral ligament (LCL) that led to their contribution to elbow contracture. In our rat model of elbow PTJC, a unilateral surgery replicated damage that commonly occurs due to elbow dislocation. Following surgery, the injured limb was immobilized for 42 days. The capsule and LCL were evaluated after 42 days of immobilization or 42 days of immobilization followed by 42 days of free mobilization. We evaluated extracellular matrix protein biochemistry, non-enzymatic collagen crosslink content, tissue volume with contrast-enhanced micro-computed tomography, and tissue mechanical properties. Increased collagen content, but not collagen density, was observed in both injured limb capsules and LCLs, which was consistent with the increased tissue volume. Injured limb LCLs exhibited decreased normalized maximum force, and both tissues had increased immature collagen cross-links compared to control. Overall, increased tissue volume and immature collagen crosslinks in the capsule and LCL drive their contribution to elbow contracture in our rat model.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jor.24781DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744303PMC
August 2021

Dysregulated assembly of elastic fibers in fibulin-5 knockout mice results in a tendon-specific increase in elastic modulus.

J Mech Behav Biomed Mater 2021 01 7;113:104134. Epub 2020 Oct 7.

Department of Biomedical Engineering, Washington University in St. Louis, USA; Department of Mechanical Engineering and Materials Science, Washington University in St. Louis, USA; Department of Orthopaedic Surgery, Washington University in St. Louis, USA. Electronic address:

Elastic fiber assembly is coordinated in part by fibulin-5, a matricellular protein. When fibulin-5 is not available to guide elastogenesis, elastin forms into disconnected globules instead of the dense elastic fiber core found in healthy tissues. Despite the growing evidence for a significant role of elastic fibers in tendon mechanics and the clinical relevance to cutis laxa, a human disease which can be caused by a mutation in the gene encoding fibulin-5, it is unknown how malformed elastic fibers affect tendon function. Therefore, this study investigated the effects of dysregulated elastic fiber assembly in tendons from fibulin-5 knockout mice in comparison to wild-type controls. Due to evidence for a more prominent role of elastic fibers in tendons with higher functional demands, both the energy-storing Achilles tendon and the more positional tibialis anterior tendon were evaluated. The linear modulus of knockout Achilles tendons was increased compared to controls, yet there was no discernible change in mechanical properties of the tibialis anterior tendon across genotypes. Transmission electron microscopy confirmed the presence of malformed elastic fibers in knockout tendons while no other changes to tendon composition or structure were found. The mechanism behind the increase in linear modulus in fibulin-5 knockout Achilles tendons may be greater collagen engagement due to decreased regulation of strain-induced structural reorganization. These findings support the theory of a significant, functionally distinct role of elastic fibers in tendon mechanics.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jmbbm.2020.104134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8146012PMC
January 2021

Minoxidil Cannot Be Used To Target Lysyl Hydroxylases during Postnatal Mouse Lung Development: A Cautionary Note.

J Pharmacol Exp Ther 2020 12 5;375(3):478-487. Epub 2020 Oct 5.

Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, member of the German Center for Lung Research (DZL), Bad Nauheim, Germany (T.P., E.L., I.M., D.E.S.S., D.M., W.S., R.E.M.); Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center (UGMLC), member of the German Center for Lung Research (DZL), Giessen, Germany (T.P., E.L., I.M., D.E.S.S., D.M., I.V., S.H., W.S., R.E.M.); Division of Regenerative Medicine, Department of Plastic and Reconstructive Surgery, St. Marianna University School of Medicine, Kawasaki, Japan (H.I.); and Institute of Virology and Cell Biology (H.S., J.B.) and Department of Dermatology (J.B.), University of Lübeck, Lübeck, Germany,

The lysyl hydroxylases (procollagen-lysine 5-dioxygenases) PLOD1, PLOD2, and PLOD3 have been proposed as pathogenic mediators of stunted lung development in bronchopulmonary dysplasia (BPD), a common complication of preterm birth. In affected infants, pulmonary oxygen toxicity stunts lung development. Mice lacking exhibit 15% mortality, and mice lacking or exhibit embryonic lethality. Therefore, to address any pathogenic role of lysyl hydroxylases in stunted lung development associated with BPD, minoxidil was administered to newborn mice in an oxygen toxicity-based BPD animal model. Minoxidil, which has attracted much interest in the management of systemic hypertension and androgenetic alopecia, can also be used to reduce lysyl hydroxylase activity in cultured cells. An in vivo pilot dosing study established 50 mg⋅kg⋅day as the maximum possible minoxidil dose for intraperitoneal administration in newborn mouse pups. When administered at 50 mg⋅kg⋅day to newborn mouse pups, minoxidil was detected in the lungs but did not impact lysine hydroxylation, collagen crosslinking, or lysyl hydroxylase expression in the lungs. Consistent with no impact on mouse lung extracellular matrix structures, minoxidil administration did not alter the course of normal or stunted lung development in newborn mice. At doses of up to 50 mg⋅kg⋅day, pharmacologically active concentrations of minoxidil were not achieved in neonatal mouse lung tissue; thus, minoxidil cannot be used to attenuate lysyl hydroxylase expression or activity during mouse lung development. These data also highlight the need for new and specific lysyl hydroxylase inhibitors. SIGNIFICANCE STATEMENT: Extracellular matrix crosslinking is mediated by lysyl hydroxylases, which generate hydroxylated lysyl residues in procollagen peptides. Deregulated collagen crosslinking is a pathogenic component of a spectrum of diseases, and thus, there is interest in validating lysyl hydroxylases as pathogenic mediators of disease and potential "druggable" targets. Minoxidil, administered at the maximum possible dose, did not inhibit lysyl hydroxylation in newborn mouse lungs, suggesting that minoxidil was unlikely to be of use in studies that pharmacologically target lysyl hydroxylation in vivo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1124/jpet.120.000138DOI Listing
December 2020

Role of collagen XII in skin homeostasis and repair.

Matrix Biol 2020 12 2;94:57-76. Epub 2020 Sep 2.

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

Skin integrity and function depends to a large extent on the composition of the extracellular matrix, which regulates tissue organization. Collagen XII is a homotrimer with short collagenous domains that confer binding to the surface of collagen I-containing fibrils and extended flexible arms, which bind to non-collagenous matrix components. Thereby, collagen XII helps to maintain collagen suprastructure and to absorb stress. Mutant or absent collagen XII leads to reduced muscle and bone strength and lax skin, whereas increased collagen XII amounts are observed in tumor stroma, scarring and fibrosis. This study aimed at uncovering in vivo mechanisms by which collagen XII may achieve these contrasting outcomes. We analyzed skin as a model tissue that contains abundant fibrils, composed of collagen I, III and V with collagen XII decorating their surface, and which is subject to mechanical stress. The impact of different collagen XII levels was investigated in collagen XII-deficient (Col12-KO) mice and in mice with collagen XII overexpression in the dermis (Col12-OE). Unchallenged skin of these mice was histologically inconspicuous, but at the ultrastructural level revealed distinct aberrations in collagen network suprastructure. Repair of excisional wounds deviated from controls in both models by delayed healing kinetics, which was, however, caused by completely different mechanisms in the two mouse lines. The disorganized matrix in Col12-KO wounds failed to properly sequester TGFβ, resulting in elevated numbers of myofibroblasts. These are, however, unable to contract and remodel the collagen XII-deficient matrix. Excess of collagen XII, in contrast, promotes persistence of M1-like macrophages in the wound bed, thereby stalling the wounds in an early inflammatory stage of the repair process and delaying healing. Taken together, we demonstrate that collagen XII is a key component that assists in orchestrating proper skin matrix structure, controls growth factor availability and regulates cellular composition and function. Together, these functions are pivotal for re-establishing homeostasis after injury.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.matbio.2020.08.002DOI Listing
December 2020

A Dual-Acting Nitric Oxide Donor and Phosphodiesterase 5 Inhibitor Promotes Wound Healing in Normal Mice and Mice with Diabetes.

J Invest Dermatol 2021 02 27;141(2):415-426. Epub 2020 Jun 27.

Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Zurich, Switzerland. Electronic address:

Chronic wounds affect a large percentage of the population worldwide and cause significant morbidity. Unfortunately, efficient compounds for the treatment of chronic wounds are yet not available. Endothelial dysfunction, which is at least in part a result of compromised nitric oxide production and concomitant reduction in cGMP levels, is a major pathologic feature of chronic wounds. Therefore, we designed and synthesized a compound with a unique dual-acting activity (TOP-N53), acting as a nitric oxide donor and phosphodiesterase 5 inhibitor, and applied it locally to full-thickness skin wounds in healthy and healing-impaired mice with diabetes. TOP-N53 promoted keratinocyte proliferation, angiogenesis, and collagen maturation in healthy mice without accelerating the wound inflammatory response or scar formation. Most importantly, it partially rescued the healing impairment of mice with genetically determined type II diabetes (db/db) by stimulating re-epithelialization and granulation tissue formation, including angiogenesis. In vitro studies with human and murine primary cells showed a positive effect of TOP-N53 on keratinocyte and fibroblast migration, keratinocyte proliferation, and endothelial cell migration and tube formation. These results demonstrate a remarkable healing-promoting activity of TOP-N53 by targeting the major resident cells in the wound tissue.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jid.2020.05.111DOI Listing
February 2021

Activin-mediated alterations of the fibroblast transcriptome and matrisome control the biomechanical properties of skin wounds.

Nat Commun 2020 05 25;11(1):2604. Epub 2020 May 25.

Institute of Molecular Health Sciences, Department of Biology, ETH Zurich, Otto-Stern-Weg 7, 8093, Zurich, Switzerland.

Matrix deposition is essential for wound repair, but when excessive, leads to hypertrophic scars and fibrosis. The factors that control matrix deposition in skin wounds have only partially been identified and the consequences of matrix alterations for the mechanical properties of wounds are largely unknown. Here, we report how a single diffusible factor, activin A, affects the healing process across scales. Bioinformatics analysis of wound fibroblast transcriptome data combined with biochemical and histopathological analyses of wounds and functional in vitro studies identify that activin promotes pro-fibrotic gene expression signatures and processes, including glycoprotein and proteoglycan biosynthesis, collagen deposition, and altered collagen cross-linking. As a consequence, activin strongly reduces the wound and scar deformability, as identified by a non-invasive in vivo method for biomechanical analysis. These results provide mechanistic insight into the roles of activin in wound repair and fibrosis and identify the functional consequences of alterations in the wound matrisome at the biomechanical level.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-020-16409-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7248062PMC
May 2020

Increased Collagen Turnover Impairs Tendon Microstructure and Stability in Integrin α2β1-Deficient Mice.

Int J Mol Sci 2020 Apr 18;21(8). Epub 2020 Apr 18.

Department of Regenerative Musculoskeletal Medicine, Institute for Musculoskeletal Medicine, Westfälische Wilhelms-University, 48149 Münster, Germany.

Integrins are a family of transmembrane proteins, involved in substrate recognition and cell adhesion in cross-talk with the extra cellular matrix. In this study, we investigated the influence of integrin α2β1 on tendons, another collagen type I-rich tissue of the musculoskeletal system. Morphological, as well as functional, parameters were analyzed in vivo and in vitro, comparing wild-type against integrin α2β1 deficiency. Tenocytes lacking integrin α2β1 produced more collagen in vitro, which is similar to the situation in osseous tissue. Fibril morphology and biomechanical strength proved to be altered, as integrin α2β1 deficiency led to significantly smaller fibrils as well as changes in dynamic E-modulus in vivo. This discrepancy can be explained by a higher collagen turnover: integrin α2β1-deficient cells produced more matrix, and tendons contained more residual C-terminal fragments of type I collagen, as well as an increased matrix metalloproteinase-2 activity. A greatly decreased percentage of non-collagenous proteins may be the cause of changes in fibril diameter regulation and increased the proteolytic degradation of collagen in the integrin-deficient tendons. The results reveal a significant impact of integrin α2β1 on collagen modifications in tendons. Its role in tendon pathologies, like chronic degradation, will be the subject of future investigations.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21082835DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7215526PMC
April 2020

A comprehensive map of human elastin cross-linking during elastogenesis.

FEBS J 2019 09 5;286(18):3594-3610. Epub 2019 Jun 5.

Fraunhofer Institute for Microstructure of Materials and Systems IMWS, Halle (Saale), Germany.

Elastin is an essential structural protein in the extracellular matrix of vertebrates. It is the core component of elastic fibers, which enable connective tissues such as those of the skin, lungs or blood vessels to stretch and recoil. This function is provided by elastin's exceptional properties, which mainly derive from a unique covalent cross-linking between hydrophilic lysine-rich motifs of units of the monomeric precursor tropoelastin. To date, elastin's cross-linking is poorly investigated. Here, we purified elastin from human tissue and cleaved it into soluble peptides using proteases with different specificities. We then analyzed elastin's molecular structure by identifying unmodified residues, post-translational modifications and cross-linked peptides by high-resolution mass spectrometry and amino acid analysis. The data revealed the presence of multiple isoforms in parallel and a complex and heterogeneous molecular interconnection. We discovered that the same lysine residues in different monomers were simultaneously involved in various cross-link types or remained unmodified. Furthermore, both types of cross-linking domains, Lys-Pro and Lys-Ala domains, participate not only in bifunctional inter- but also in intra-domain cross-links. We elucidated the sequences of several desmosine-containing peptides and the contribution of distinct domains such as 6, 14 and 25. In contrast to earlier assumptions proposing that desmosine cross-links are formed solely between two domains, we elucidated the structure of a peptide that proves a desmosine formation with participation of three Lys-Ala domains. In summary, these results provide new and detailed insights into the cross-linking process, which takes place within and between human tropoelastin units in a stochastic manner.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.14929DOI Listing
September 2019

Elastin is heterogeneously cross-linked.

J Biol Chem 2018 09 14;293(39):15107-15119. Epub 2018 Aug 14.

From the Institute of Pharmacy, Martin Luther University Halle-Wittenberg, Halle (Saale) 06120, Germany,

Elastin is an essential vertebrate protein responsible for the elasticity of force-bearing tissues such as those of the lungs, blood vessels, and skin. One of the key features required for the exceptional properties of this durable biopolymer is the extensive covalent cross-linking between domains of its monomer molecule tropoelastin. To date, elastin's exact molecular assembly and mechanical properties are poorly understood. Here, using bovine elastin, we investigated the different types of cross-links in mature elastin to gain insight into its structure. We purified and proteolytically cleaved elastin from a single tissue sample into soluble cross-linked and noncross-linked peptides that we studied by high-resolution MS. This analysis enabled the elucidation of cross-links and other elastin modifications. We found that the lysine residues within the tropoelastin sequence were simultaneously unmodified and involved in various types of cross-links with different other domains. The Lys-Pro domains were almost exclusively linked via lysinonorleucine, whereas Lys-Ala domains were found to be cross-linked via lysinonorleucine, allysine aldol, and desmosine. Unexpectedly, we identified a high number of intramolecular cross-links between lysine residues in close proximity. In summary, we show on the molecular level that elastin formation involves random cross-linking of tropoelastin monomers resulting in an unordered network, an unexpected finding compared with previous assumptions of an overall beaded structure.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.RA118.004322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166741PMC
September 2018

Roles of fibronectin isoforms in neonatal vascular development and matrix integrity.

PLoS Biol 2018 07 23;16(7):e2004812. Epub 2018 Jul 23.

Faculty of Medicine, Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada.

Fibronectin (FN) exists in two forms-plasma FN (pFN) and cellular FN (cFN). Although the role of FN in embryonic blood vessel development is well established, its function and the contribution of individual isoforms in early postnatal vascular development are poorly understood. Here, we employed a tamoxifen-dependent cFN inducible knockout (cFN iKO) mouse model to study the consequences of postnatal cFN deletion in smooth muscle cells (SMCs), the major cell type in the vascular wall. Deletion of cFN influences collagen deposition but does not affect life span. Unexpectedly, pFN translocated to the aortic wall in the cFN iKO and in control mice, possibly rescuing the loss of cFN. Postnatal pFN deletion did not show a histological aortic phenotype. Double knockout (dKO) mice lacking both, cFN in SMCs and pFN, resulted in postnatal lethality. These data demonstrate a safeguard role of pFN in vascular stability and the dispensability of the individual FN isoforms in postnatal vascular development. Complete absence of FNs in the dKOs resulted in a disorganized tunica media of the aortic wall. Matrix analysis revealed common and differential roles of the FN isoforms in guiding the assembly/deposition of elastogenic extracellular matrix (ECM) proteins in the aortic wall. In addition, we determined with two cell culture models that that the two FN isoforms acted similarly in supporting matrix formation with a greater contribution from cFN. Together, these data show that pFN exerts a critical role in safeguarding vascular organization and health, and that the two FN isoforms function in an overlapping as well as distinct manner to maintain postnatal vascular matrix integrity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.2004812DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6072322PMC
July 2018

Nrf2-Mediated Fibroblast Reprogramming Drives Cellular Senescence by Targeting the Matrisome.

Dev Cell 2018 07;46(2):145-161.e10

Institute of Molecular Health Sciences, Department of Biology, Swiss Federal Institute of Technology (ETH) Zurich, Otto-Stern-Weg 7, 8093 Zurich, Switzerland. Electronic address:

Nrf2 is a key regulator of the antioxidant defense system, and pharmacological Nrf2 activation is a promising strategy for cancer prevention and promotion of tissue repair. Here we show, however, that activation of Nrf2 in fibroblasts induces cellular senescence. Using a combination of transcriptomics, matrix proteomics, chromatin immunoprecipitation and bioinformatics we demonstrate that fibroblasts with activated Nrf2 deposit a senescence-promoting matrix, with plasminogen activator inhibitor-1 being a key inducer of the senescence program. In vivo, activation of Nrf2 in fibroblasts promoted re-epithelialization of skin wounds, but also skin tumorigenesis. The pro-tumorigenic activity is of general relevance, since Nrf2 activation in skin fibroblasts induced the expression of genes characteristic for cancer-associated fibroblasts from different mouse and human tumors. Therefore, activated Nrf2 qualifies as a marker of the cancer-associated fibroblast phenotype. These data highlight the bright and the dark sides of Nrf2 and the need for time-controlled activation of this transcription factor.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.devcel.2018.06.012DOI Listing
July 2018

Targeting transglutaminase 2 partially restores extracellular matrix structure but not alveolar architecture in experimental bronchopulmonary dysplasia.

FEBS J 2018 08 7;285(16):3056-3076. Epub 2018 Jul 7.

Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Member of the German Center for Lung Research (DZL), Giessen, Germany, Bad Nauheim, Germany.

The generation, maturation and remodelling of the extracellular matrix (ECM) are essential for the formation of alveoli during lung development. Alveoli formation is disturbed in preterm infants that develop bronchopulmonary dysplasia (BPD), where collagen fibres are malformed, and perturbations to lung ECM structures may underlie BPD pathogenesis. Malformed ECM structures might result from abnormal protein cross-linking, in part attributable to the increased expression and activity of transglutaminase 2 (TGM2) that have been noted in affected patient lungs, as well as in hyperoxia-based BPD animal models. The objective of the present study was to assess whether TGM2 plays a causal role in normal and aberrant lung alveolarization. Targeted deletion of Tgm2 in C57BL/6J mice increased septal thickness and reduced gas-exchange surface area in otherwise normally developing lungs. During aberrant lung alveolarization that occurred under hyperoxic conditions, collagen structures in Tgm2 mice were partially protected from the impact of hyperoxia, where normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance was restored; however, the lung alveolar architecture remained abnormal. Inhibition of transglutaminases (including TGM2) with cysteamine appreciably reduced transglutaminase activity in vivo, as assessed by N -(γ-l-glutamyl)-l-lysine abundance and TGM catalytic activity, and restored normal dihydroxylysinonorleucine and hydroxylysylpiridinoline collagen cross-link abundance under pathological conditions. Furthermore, a moderate improvement in alveoli size and gas-exchange surface density was noted in cysteamine-treated mouse lungs in which BPD was modelled. These data indicate that TGM2 plays a role in normal lung alveolarization, and contributes to the formation of aberrant ECM structures during disordered lung alveolarization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/febs.14596DOI Listing
August 2018

A type VII collagen subdomain mutant is thermolabile and shows enhanced proteolytic degradability - Implications for the pathogenesis of recessive dystrophic epidermolysis bullosa?

Biochim Biophys Acta Mol Basis Dis 2017 01 28;1863(1):52-59. Epub 2016 Oct 28.

Institute of Chemistry, University of Lübeck, Ratzeburger Allee 160, 23562 Lübeck, Germany. Electronic address:

Type VII collagen is the major constituent of anchoring fibrils. It has a central collagenous domain that is surrounded by a small C-terminal non-collagenous domain (NC2) and a large N-terminal non-collagenous (NC1) domain. Mutations in type VII collagen can lead to hereditary skin blistering disease dystrophic epidermolysis bullosa (DEB). Most of the pathogenic missense mutations are within the collagenous domain. NC1 domain mediates interactions with other extracellular matrix molecules and only very few missense mutations within NC1 causing DEB have been reported. Interestingly, fibronectin III like (FNIII) domain 8 in the human protein can harbour different mutations at position 886 with one (R886P) leading to recessive DEB, whereas the others do not. We characterized subdomains of murine NC1, the FNIII domains 7-8, and the individual domains FNIII7 and FNIII8 by NMR- and CD-spectroscopy. We analysed the influence on stability for a mutation causing DEB and a non-pathogenic mutation. Whereas the silent mutation behaves as the wild type, the pathogenic mutation leads to a dramatic decrease in thermal stability of the FNIII8 domain. The melting temperature lowered from 77°C to 40°C compared to the wild type protein. This renders the domain susceptible to protease cleavage which could be shown by degradation tests with cathepsin G, cathepsin K, and MMP9. Our data show partial unfolding of type VII collagen due to the mutation causes an increased degradation. This could lead to skin blistering and opens new concomitant treatment options in some types of type VII collagen related skin blistering diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbadis.2016.10.023DOI Listing
January 2017

Fibroblast-Derived MMP-14 Regulates Collagen Homeostasis in Adult Skin.

J Invest Dermatol 2016 08 9;136(8):1575-1583. Epub 2016 Apr 9.

Department of Dermatology and Venerology, University of Cologne, Cologne, Germany.

Proteolytic activities in the extracellular matrix by the matrix metalloproteinase (MMP)-14 have been implicated in the remodeling of collagenous proteins during development. To analyze the function of fibroblast-derived MMP-14 in adult skin homeostasis, we generated mice with inducible deletion of MMP-14 in the dermal fibroblast (MMP-14(Sf-/-)). These mice are smaller and display a fibrosis-like phenotype in the skin. The skin of these mice showed increased stiffness and tensile strength but no altered collagen cross-links. In vivo, we measured a significantly increased amount of collagen type I accumulated in the skin of MMP-14(Sf-/-) mice without an increase in collagen fibril diameters. However, bleomycin-induced fibrosis in skin proceeded in a comparable manner in MMP-14(Sf+/+) and MMP-14(Sf-/-) mice, but resolution over time was impaired in MMP-14(Sf-/-) mice. Increased accumulation of collagen type I was detected in MMP-14(Sf-/-) fibroblasts in culture without significant enhancement of collagen de novo synthesis. This points to a degradative but not synthetic phenotype. In support of this, MMP-14(Sf-/-) fibroblasts lost their ability to process fibrillar collagen type I and to activate proMMP-2. Taken together, these data indicate that MMP-14 expression in fibroblasts plays a crucial role in collagen remodeling in adult skin and largely contributes to dermal homeostasis underlying its pathogenic role in fibrotic skin disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jid.2016.03.036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4946792PMC
August 2016

The Extracellular Matrix Signature in Vein Graft Disease.

Can J Cardiol 2016 08 1;32(8):1008.e11-7. Epub 2015 Dec 1.

Department of Dermatology, University of Lübeck, Lübeck, Germany; Institute of Virology and Cell Biology, University of Lübeck, Lübeck, Germany. Electronic address:

Background: Vein graft disease is a major and yet unsolved problem in cardiac revascularization surgery. Although accumulation of extracellular matrix is characteristic for vein graft disease, detailed analysis of the fibrotic material is lacking. Because alterations of collagen cross-links are typical for organ fibrosis, we performed a comprehensive analysis of collagen and elastin in vein graft disease.

Methods: Collagen, elastin, and their respective cross-links were analyzed using histology and amino acid analysis. The expression of collagen-modifying enzymes was analyzed using SYBR Green quantitative real-time polymerase chain reaction. Fibrillin expression was analyzed by immunohistochemistry and quantitative real-time polymerase chain reaction.

Results: Diseased vein grafts showed a marked increase of collagen and of intermediate collagen cross-links, which are markers for newly synthesized collagen. Furthermore, we identified in vein graft disease increased levels of mature hydroxylysine aldehyde-derived cross-links typical for skeletal tissues. This was accompanied by upregulation of lysyl hydroxylase 2 and lysyl oxidase expression. Furthermore, vein graft disease showed a reduction of the elastin/collagen ratio, using elastin cross-links as a marker of elastin content, which was accompanied by an increase of fibrillin-1.

Conclusions: Vein graft disease was accompanied by marked alterations in the composition of the extracellular matrix. The altered collagen cross-link pattern and the reduced elastin/collagen ratio might synergistically increase the stiffness in diseased vein grafts. Furthermore, hydroxylysine aldehyde-derived cross-links can cause a decreased degradability of collagens by matrix-metalloproteinases. Our data suggest collagen cross-links as a therapeutic target in vein graft disease.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cjca.2015.11.014DOI Listing
August 2016

COMP-assisted collagen secretion--a novel intracellular function required for fibrosis.

J Cell Sci 2016 Feb 8;129(4):706-16. Epub 2016 Jan 8.

Department of Dermatology, University of Cologne, Cologne, Germany

Cartilage oligomeric matrix protein (COMP) is an abundant component in the extracellular matrix (ECM) of load-bearing tissues such as tendons and cartilage. It provides adaptor functions by bridging different ECM structures. We have previously shown that COMP is also a constitutive component of healthy human skin and is strongly induced in fibrosis. It binds directly and with high affinity to collagen I and to collagen XII that decorates the surface of collagen I fibrils. We demonstrate here that lack of COMP-collagen interaction in the extracellular space leads to changes in collagen fibril morphology and density, resulting in altered skin biomechanical properties. Surprisingly, COMP also fulfills an important intracellular function in assisting efficient secretion of collagens, which were retained in the endoplasmic reticulum of COMP-null fibroblasts. Accordingly, COMP-null mice showed severely attenuated fibrotic responses in skin. Collagen secretion was fully restored by introducing wild-type COMP. Hence, our work unravels a new, non-structural and intracellular function of the ECM protein COMP in controlling collagen secretion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.180216DOI Listing
February 2016

Interleukin-4 Receptor α Signaling in Myeloid Cells Controls Collagen Fibril Assembly in Skin Repair.

Immunity 2015 Oct;43(4):803-16

Department of Dermatology, University of Cologne, 50937 Cologne, Germany; Center for Molecular Medicine Cologne, University of Cologne, 50931 Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, 50674 Cologne, Germany. Electronic address:

Activation of the immune response during injury is a critical early event that determines whether the outcome of tissue restoration is regeneration or replacement of the damaged tissue with a scar. The mechanisms by which immune signals control these fundamentally different regenerative pathways are largely unknown. We have demonstrated that, during skin repair in mice, interleukin-4 receptor α (IL-4Rα)-dependent macrophage activation controlled collagen fibril assembly and that this process was important for effective repair while having adverse pro-fibrotic effects. We identified Relm-α as one important player in the pathway from IL-4Rα signaling in macrophages to the induction of lysyl hydroxylase 2 (LH2), an enzyme that directs persistent pro-fibrotic collagen cross-links, in fibroblasts. Notably, Relm-β induced LH2 in human fibroblasts, and expression of both factors was increased in lipodermatosclerosis, a condition of excessive human skin fibrosis. Collectively, our findings provide mechanistic insights into the link between type 2 immunity and initiation of pro-fibrotic pathways.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.immuni.2015.09.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4681399PMC
October 2015

Collagen and elastin cross-linking is altered during aberrant late lung development associated with hyperoxia.

Am J Physiol Lung Cell Mol Physiol 2015 Jun 3;308(11):L1145-58. Epub 2015 Apr 3.

Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany; Department of Internal Medicine (Pulmonology), University of Giessen and Marburg Lung Center, German Center for Lung Research, Giessen, Germany;

Maturation of the lung extracellular matrix (ECM) plays an important role in the formation of alveolar gas exchange units. A key step in ECM maturation is cross-linking of collagen and elastin, which imparts stability and functionality to the ECM. During aberrant late lung development in bronchopulmonary dysplasia (BPD) patients and animal models of BPD, alveolarization is blocked, and the function of ECM cross-linking enzymes is deregulated, suggesting that perturbed ECM cross-linking may impact alveolarization. In a hyperoxia (85% O2)-based mouse model of BPD, blunted alveolarization was accompanied by alterations to lung collagen and elastin levels and cross-linking. Total collagen levels were increased (by 63%). The abundance of dihydroxylysinonorleucine collagen cross-links and the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio were increased by 11 and 18%, respectively, suggestive of a profibrotic state. In contrast, insoluble elastin levels and the abundance of the elastin cross-links desmosine and isodesmosine in insoluble elastin were decreased by 35, 30, and 21%, respectively. The lung collagen-to-elastin ratio was threefold increased. Treatment of hyperoxia-exposed newborn mice with the lysyl oxidase inhibitor β-aminopropionitrile partially restored normal collagen levels, normalized the dihydroxylysinonorleucine-to-hydroxylysinonorleucine ratio, partially normalized desmosine and isodesmosine cross-links in insoluble elastin, and partially restored elastin foci structure in the developing septa. However, β-aminopropionitrile administration concomitant with hyperoxia exposure did not improve alveolarization, evident from unchanged alveolar surface area and alveoli number, and worsened septal thickening (increased by 12%). These data demonstrate that collagen and elastin cross-linking are perturbed during the arrested alveolarization of developing mouse lungs exposed to hyperoxia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1152/ajplung.00039.2015DOI Listing
June 2015

Lysyl oxidases play a causal role in vascular remodeling in clinical and experimental pulmonary arterial hypertension.

Arterioscler Thromb Vasc Biol 2014 Jul 15;34(7):1446-58. Epub 2014 May 15.

From the Division of Pulmonology, Department of Internal Medicine, University of Giessen and Marburg Lung Center (UGMLC), Giessen, Germany (A.H.N., I.M., G.N., F.R., M.L.T., F.V., S.H., K.M., I.V., N.W., W.S., R.E.M.); Department of Lung Development and Remodelling, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany (A.H.N., I.M., G.N., W.S., R.E.M.); and the Department of Dermatology (J.B.) and Institute of Virology and Cell Biology (J.B., H.S.), University of Lübeck, Lübeck, Germany.

Objective: Pulmonary vascular remodeling, the pathological hallmark of pulmonary arterial hypertension, is attributed to proliferation, apoptosis resistance, and migration of vascular cells. A role of dysregulated matrix cross-linking and stability as a pathogenic mechanism has received little attention. We aimed to assess whether matrix cross-linking enzymes played a causal role in experimental pulmonary hypertension (PH).

Approach And Results: All 5 lysyl oxidases were detected in concentric and plexiform vascular lesions of patients with idiopathic pulmonary arterial hypertension. Lox, LoxL1, LoxL2, and LoxL4 expression was elevated in lungs of patients with idiopathic pulmonary arterial hypertension, whereas LoxL2 and LoxL3 expression was elevated in laser-capture microdissected vascular lesions. Lox expression was hypoxia-responsive in pulmonary artery smooth muscle cells and adventitial fibroblasts, whereas LoxL1 and LoxL2 expression was hypoxia-responsive in adventitial fibroblasts. Lox expression was increased in lungs from hypoxia-exposed mice and in lungs and pulmonary artery smooth muscle cells of monocrotaline-treated rats, which developed PH. Pulmonary hypertensive mice exhibited increased muscularization and perturbed matrix structures in vessel walls of small pulmonary arteries. Hypoxia exposure led to increased collagen cross-linking, by dihydroxylysinonorleucine and hydroxylysinonorleucine cross-links. Administration of the lysyl oxidase inhibitor β-aminopropionitrile attenuated the effect of hypoxia, limiting perturbations to right ventricular systolic pressure, right ventricular hypertrophy, and vessel muscularization and normalizing collagen cross-linking and vessel matrix architecture.

Conclusions: Lysyl oxidases are dysregulated in clinical and experimental PH. Lysyl oxidases play a causal role in experimental PH and represent a candidate therapeutic target. Our proof-of-principle study demonstrated that modulation of lung matrix cross-linking can affect pulmonary vascular remodeling associated with PH.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1161/ATVBAHA.114.303534DOI Listing
July 2014

Genetic ablation of mast cells redefines the role of mast cells in skin wound healing and bleomycin-induced fibrosis.

J Invest Dermatol 2014 Jul 9;134(7):2005-2015. Epub 2014 Jan 9.

Department of Dermatology, University of Cologne, Cologne, Germany; Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany; Center for Molecular Medicine (CMMC), University of Cologne, Cologne, Germany. Electronic address:

Conclusive evidence for the impact of mast cells (MCs) in skin repair is still lacking. Studies in mice examining the role of MC function in the physiology and pathology of skin regenerative processes have obtained contradictory results. To clarify the specific role of MCs in regenerative conditions, here we used a recently developed genetic mouse model that allows conditional MC ablation to examine MC-specific functions in skin. This mouse model is based on the cell type-specific expression of Cre recombinase in connective tissue-type MCs under control of the Mcpt5 promoter and the Cre-inducible diphtheria toxin receptor-mediated cell lineage ablation by diphtheria toxin. In response to excisional skin injury, genetic ablation of MCs did not affect the kinetics of reepithelialization, the formation of vascularized granulation tissue, or scar formation. Furthermore, genetic ablation of MCs failed to prevent the development of skin fibrosis upon bleomycin challenge. The amount of deposited collagen and the biochemistry of collagen fibril crosslinks within fibrotic lesions were comparable in MC-depleted and control mice. Collectively, our findings strongly suggest that significant reduction of MC numbers does not affect skin wound healing and bleomycin-induced fibrosis in mice, and provide to our knowledge previously unreported insight in the long-debated contribution of MCs in skin regenerative processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2014.12DOI Listing
July 2014

TSG-6 released from intradermally injected mesenchymal stem cells accelerates wound healing and reduces tissue fibrosis in murine full-thickness skin wounds.

J Invest Dermatol 2014 Feb 6;134(2):526-537. Epub 2013 Aug 6.

Department of Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany. Electronic address:

Proper activation of macrophages (Mφ) in the inflammatory phase of acute wound healing is essential for physiological tissue repair. However, there is a strong indication that robust Mφ inflammatory responses may be causal for the fibrotic response always accompanying adult wound healing. Using a complementary approach of in vitro and in vivo studies, we here addressed the question of whether mesenchymal stem cells (MSCs)-due to their anti-inflammatory properties-would control Mφ activation and tissue fibrosis in a murine model of full-thickness skin wounds. We have shown that the tumor necrosis factor-α (TNF-α)-stimulated protein 6 (TSG-6) released from MSCs in co-culture with activated Mφ or following injection into wound margins suppressed the release of TNF-α from activated Mφ and concomitantly induced a switch from a high to an anti-fibrotic low transforming growth factor-β1 (TGF-β1)/TGF-β3 ratio. This study provides insight into what we believe to be a previously undescribed multifaceted role of MSC-released TSG-6 in wound healing. MSC-released TSG-6 was identified to improve wound healing by limiting Mφ activation, inflammation, and fibrosis. TSG-6 and MSC-based therapies may thus qualify as promising strategies to enhance tissue repair and to prevent excessive tissue fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/jid.2013.328DOI Listing
February 2014

Influence of silicone gel on standardized postoperative scars.

J Dtsch Dermatol Ges 2013 May 5;11(5):412-9. Epub 2013 Feb 5.

Department of General Surgery, Sana Clinics Eastern Holstein, Clinic Eutin, Germany.

Background: Silicone gel is one therapeutic approach in the treatment and prevention of excessive scarring. The likely mechanism of action is the hydration of the tissue. This should lead to reduced angiogenesis and capillary blood flow. The efficacy is still controversial and the evidence base, insufficient. The aim of this prospective and standardized study is to investigate silicone gel in the preventive treatment of scars.

Patients And Methods: Included in the study were 20 patients with costal cartilage harvest. Half of a standard chest scar was treated for three months with a silicone gel. The other half served as an internal control. After three months both scar sides were compared subjectively by visual analog scale and objectively by elasticity, moisture and color measurements.

Results: Of 19 patients 8 had a better subjective result in the treated half. In one subject, no difference was seen. A worse subjective result in the treated half was seen in 10 out of 19. The objective measurements showed no significant difference. A correlation between the different results was not seen.

Conclusions: The use of silicone gel caused subjective differences within the same scar (worsening as well as improvement of the appearance). Positive effects were not detectable in the investigated parameters.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/ddg.12011DOI Listing
May 2013

Elevated MMP-7 levels in patients with systemic sclerosis: correlation with pulmonary involvement.

Exp Dermatol 2011 Sep 26;20(9):770-3. Epub 2011 Jun 26.

Background: Fibrosis is characterized by an excessive accumulation of connective tissue because of an imbalance between synthesis and degradation of extracellular matrix proteins. Systemic sclerosis (SSc) is a prototypic chronic inflammatory disease leading to a severe fibrosis of the skin and many internal organs. QUESTIONS ADDRESSED: We investigated whether serum MMP-7 levels reflect the activity of the fibrotic reaction in systemic sclerosis.

Experimental Design: Serum samples were obtained from 123 patients with systemic sclerosis. MMP-serum levels of all patients with SSc were compared with age-matched healthy controls.

Results: Significantly increased median serum MMP-7 levels were found in patients with SSc when compared with controls. The median MMP-7 serum level of patients with lung fibrosis (LF) was significantly higher compared with those without LF. Accordingly, patients with dyspnea and DLCO (diffusion capacity of the lung for carbon monoxide) levels below 60% showed significantly higher median MMP-7 levels.

Conclusions: Elevated MMP-7 levels are associated with an advanced stage of SSc and LF. These data suggest that in SSc MMP-7 is involved in the process of fibrotic tissue remodelling.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1600-0625.2011.01321.xDOI Listing
September 2011

Enhanced fibrillin-2 expression is a general feature of wound healing and sclerosis: potential alteration of cell attachment and storage of TGF-beta.

Lab Invest 2010 May 1;90(5):739-52. Epub 2010 Mar 1.

Department of Dermatology, University of Lübeck, Lübeck, Germany.

Wound healing and sclerosis are characterized by an increase of extracellular matrix proteins, which are characteristically expressed in the embryo-fetal period. We analyzed the expression of fibrillin-2, which is typically found in embryonic tissues, but only scarcely in adult skin. In wound healing and sclerotic skin diseases such as lipodermatosclerosis and scleroderma, a marked increase of fibrillin-2 expression was found by immunohistology. Double labelling of fibrillin-2 and tenascin-C, which is also expressed in wound healing and sclerosis, showed co-localization of both proteins. Solid-phase and slot blot-overlay assays showed a dose-dependent binding of the recombinant N-terminal half of fibrillin-2 (rFBN2-N) to tenascin-C. Real-time PCR showed an increase of the fibrillin-2 gene expression in cell culture triggered by typical mediators for fibroblast activation such as serum, IL-4, and TGF-beta. By contrast, prolonged hypoxia is not associated with changes in fibrillin-2 expression. Tenascin-C is an anti-adhesive substrate for fibroblasts, whereas fibrillin-2 stimulates cell attachment. Attachment assays using mixed substrates showed decreased cell attachment when tenascin-C and rFBN2-N were coated together, compared with the attachment to rFBN2-N alone. Fibrillins are involved in storage and activation of TGF-beta. Immunohistology with an antibody against the latency-associated peptide (LAP (TGF-beta1)) showed a marked increase of inactive LAP-bound TGF-beta1 in wound healing and sclerotic skin whereas normal skin showed only a weak expression. Double immunofluorescence confirmed a partial colocalization of both proteins. In conclusion, we show that a stimulation of the fibrillin-2 expression is a characteristic feature of fibroblasts present in wound healing and sclerosis, which may be involved in the alteration of cell attachment and storage of inactive TGF-beta in the matrix.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/labinvest.2010.49DOI Listing
May 2010

Alternate trafficking of cathepsin L in dermal fibroblasts induced by UVA radiation.

Exp Dermatol 2010 Aug;19(8):e117-23

Department of Dermatology, University of Cologne, Cologne, Germany.

UVA radiation is increasingly used to treat fibrotic skin disorders. However, the mechanisms underlying the therapeutic effects of UVA for these disorders are only partially understood. Cathepsin L is a lysosomal cysteine protease, which has been shown to degrade various matrix proteins thus contributing to extracellular remodeling. Therefore, we investigated whether UVA irradiation regulates the expression and release of cathepsin L in human dermal fibroblasts. No alterations were found after single irradiation; however, a significantly increased extracellular release of cathepsin L was observed after repeated irradiation up to four times. The transcript levels of cathepsin L were elevated after repetitive irradiation, leading to increased amounts of total cathepsin L protein. Furthermore, higher amounts of extracellular cathepsin L were associated with a significant reduction of intracellular processed cathepsin L and an accumulation of unprocessed procathepsin L. The use of specific inhibitors elucidated mannose phosphate-independent sorting pathways of cathepsin L leading to enhanced secretion and reduced intracellular processing. This is the first study which demonstrates that alternate trafficking mechanisms mediate the extracellular release of a cysteine protease induced by repetitive UVA irradiation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1600-0625.2009.01014.xDOI Listing
August 2010

Immunophenotyping of the human bulge region: the quest to define useful in situ markers for human epithelial hair follicle stem cells and their niche.

Exp Dermatol 2008 Jul;17(7):592-609

Department of Molecular Medicine, Max-Planck-Institute for Biochemistry, Martinsried, Germany.

Since the discovery of epithelial hair follicle stem cells (eHFSCs) in the bulge of human hair follicles (HFs) an important quest has started: to define useful markers. In the current study, we contribute to this by critically evaluating corresponding published immunoreactivity (IR) patterns, and by attempting to identify markers for the in situ identification of human eHFSCs and their niche. For this, human scalp skin cryosections of at least five different individuals were examined, employing standard immunohistology as well as increased sensitivity methods. Defined reference areas were compared by quantitative immunohistochemistry for the relative intensity of their specific IR. According to our experience, the most useful positive markers for human bulge cells turned out to be cytokeratin 15, cytokeratin 19 and CD200, but were not exclusive, while beta1 integrin and Lhx2 IR were not upregulated by human bulge keratinocytes. Absent IR for CD34, connexin43 and nestin on human bulge cells may be exploited as negative markers. alpha6 integrin, fibronectin, nidogen, fibrillin-1 and latent transforming growth factor (TGF)-beta-binding protein-1 were expressed throughout the connective tissue sheath of human HFs. On the other hand, tenascin-C was upregulated in the bulge and may thus constitute a component of the bulge stem cell niche of human HFs. These immunophenotyping results shed further light on the in situ expression patterns of claimed follicular 'stem cell markers' and suggest that not a single marker alone but only the use of a limited corresponding panel of positive and negative markers may offer a reasonable and pragmatic compromise for identifying human bulge stem cells in situ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/j.1600-0625.2008.00720.xDOI Listing
July 2008

Fibrillin-1 interactions with fibulins depend on the first hybrid domain and provide an adaptor function to tropoelastin.

J Biol Chem 2007 Mar 25;282(12):8935-46. Epub 2007 Jan 25.

Faculty of Medicine, Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada.

Fibrillin-containing microfibrils in elastic and nonelastic extracellular matrices play important structural and functional roles in various tissues, including blood vessels, lung, skin, and bone. Microfibrils are supramolecular aggregates of several protein and nonprotein components. Recently, a large region in the N-terminal portion of fibrillin-1 was characterized as a multifunctional protein interaction site, including binding sites for fibulin-2 and -5 among others. Using a panel of recombinant fibrillin-1 swapped domain and deletion fragments, we demonstrate here that the conserved first hybrid domain in fibrillin-1 is essential for binding to fibulin-2, -4, and -5. Fibulin-3 and various isoforms of fibulin-1 did not interact with fibrillin-1. Although the first hybrid domain in fibrillin-1 is located in close vicinity to the self-assembly epitope, binding of fibulin-2, -4, and -5 did not interfere with self-assembly. However, these fibulins can associate with microfibrils at various levels of maturity. Formation of ternary complexes between fibrillin-1, fibulins, and tropoelastin demonstrated that fibulin-2 and -5 but much less fibulin-4, are able to act as molecular adaptors between fibrillin-1 and tropoelastin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M608204200DOI Listing
March 2007

Functional diversity of lysyl hydroxylase 2 in collagen synthesis of human dermal fibroblasts.

Exp Cell Res 2006 Nov 28;312(18):3485-94. Epub 2006 Jul 28.

Department of Dermatology, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.

The pathogenesis of fibrosis, especially involving post-translational modifications of collagen, is poorly understood. Lysyl hydroxylase 2 (long) (LH2 (long)) is thought to play a pivotal role in fibrosis by directing the collagen cross-link pattern. Here we show that LH2 (long) exerts a bimodal function on collagen synthesis in human dermal fibroblasts. Adenoviral-mediated overexpression of LH2 (long) resulted in a mRNA increase of collagen alpha1(I) but not of fibronectin and fibrillin-1. This was accompanied by a higher mRNA level of prolyl-4-hydroxylase but not of other ER proteins (Bip, Hsp47, LH1, LH3). The collagen mRNA increase led to an elevated collagen synthesis, which was higher in the fraction of extracellularly deposited, cell-associated collagen than in the medium. The cross-link pattern of cell-associated collagen showed an increase of the hydroxylysine-aldehyde-derived cross-link dihydroxylysinonorleucine and a decrease of the lysine-aldehyde-derived component hydroxylysinonorleucine. The helical lysyl hydroxylation of the procollagen molecule was unaltered. The increase of collagen synthesis in fibroblasts overexpressing LH2 (long) was independent from cross-linking as it was also observed in the presence of beta-aminopropionitril, a cross-linking inhibitor. Together our data identify LH2 (long) as a bifunctional protein and underscores its potential role in the pathogenesis of fibrosis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yexcr.2006.07.013DOI Listing
November 2006
-->