Thorsten Steinberg

Elevated gene expression of MMP-1, MMP-10, and TIMP-1 reveal changes of molecules involved in turn-over of extracellular matrix in cyclosporine-induced gingival overgrowth.

In humans, pathogenesis in cyclosporine A (CsA)-induced gingival overgrowth (GO) includes the accumulation of extracellular matrix (ECM) constituents, viz., collagen type-1 and type-3 and proteoglycans, in subgingival connective tissue. However, whether this increase is associated with alterations of molecules pivotal for the turn-over of collagens and proteoglycans remains unclear. The present study explores the status of matrix metalloproteinase MMP-1 and MMP-10, which are important for fibrillar collagen and proteoglycan turn-over, and their tissue inhibitor TIMP-1, on their gene expression and protein levels in frozen sections derived from GO and matched normal tissue. In situ hybridization (ISH) revealed elevated levels of MMP-1 gene expression in the connective tissue of GO compared with normal tissue. This elevation also applied to MMP-10 and TIMP-1, the latter exhibiting the strongest gene transcription in the deep connective tissue. These differences detected by ISH were corroborated by quantitative reverse transcription/polymerase chain reaction; relative gene expression analysis indicated a 1.9-fold increase for MMP-1, a 2.3-fold increase for MMP-10, and a 4.8-fold increase for TIMP-1. Detection of the protein by indirect immunofluorescence showed that normal gingival tissue was devoid of all three proteins, although they were detectable in GO tissue, with emphasis on TIMP-1. Analysis of our data indicates elevated levels of MMP-1 and-10, and particularly TIMP-1. With respect to TIMP-1, this elevation may in turn lead to alterations in ECM turn-over by abrogating MMP-1 and MMP-10, thereby contributing to ECM accumulation associated with GO.

Elevated expression of genes assigned to NF-κB and apoptotic pathways in human periodontal ligament fibroblasts following mechanical stretch

There is growing evidence that apoptosis involves the nuclear transcription factor NF-κB in conjunction with related genes. However, in the context of mechanical orthodontic forces, force-sensing target genes assigned to pathways of NF-κB and apoptosis have not been fully characterised. To contribute to the identification of putative target genes, we used cDNA arrays specific for NF-κB and apoptotic pathways and analysed elevated gene expression in primary human periodontal ligament fibroblasts (PDL-F) after a 6 h application of mechanical force. Among several identified genes (including several caspases), interleukin-1β (IL-1β) and NF-κB displayed significantly higher expression on the NF-κB array, whereas higher expression was obtained for BCL2-antagonist of cell death (BAD), member 6 of the TNF-receptor superfamily (FAS) and CASP2 and RIPK1 domain-containing adaptor with death domain (CRADD) on the apoptosis array. Based on a defined cut-off level of a more than 1.5-fold higher expression, this significance in elevated gene expression was corroborated by reverse transcription/polymerase chain reaction (RT-PCR). Here, semi-quantitative (sq) PCR revealed a more pronounced elevation of mRNA gene expression in PDL-F after 6xa0h of stretch, when compared with 12xa0h. Moreover, the elevation after 6xa0h as observed by sq-PCR was convergent with quantitative PCR (q-PCR). q-PCR yielded levels of 5.8-fold higher relative gene expression for IL-1β and 1.7-fold for NF-κB, whereas that computed for BAD indicated a 5.2-fold, for CRADD a 2.1-fold and for FAS a 2.0-fold higher expression. The data obtained from the expression analysis thus indicate a stretch-induced transcriptional elevation of genes assigned to the NF-κB and apoptotic pathways. This elevation may render them target candidates for being addressed by mechanical orthodontic forces.

Reconstructing protein networks of epithelial differentiation from histological sections

MOTIVATIONnFor systems biology of complex stratified epithelia like human epidermis, it will be of particular importance to reconstruct the spatiotemporal gene and protein networks regulating keratinocyte differentiation and homeostasis.nnnRESULTSnInside the epidermis, the differentiation state of individual keratinocytes is correlated with their respective distance from the connective tissue. We here present a novel method to profile this correlation for multiple epithelial protein biomarkers in the form of quantitative spatial profiles. Profiles were computed by applying image processing algorithms to histological sections stained with tri-color indirect immunofluorescence. From the quantitative spatial profiles, reflecting the spatiotemporal changes of protein expression during cellular differentiation, graphs of protein networks were reconstructed.nnnCONCLUSIONnSpatiotemporal networks can be used as a means for comparing and interpreting quantitative spatial protein expression profiles obtained from different tissue samples. In combination with automated microscopes, our new method supports the large-scale systems biological analysis of stratified epithelial tissues.

Nuclear staining and relative distance for quantifying epidermal differentiation in biomarker expression profiling

BackgroundThe epidermal physiology results from a complex regulated homeostasis of keratinocyte proliferation, differentiation and death and is tightly regulated by a specific protein expression during cellular maturation. Cellular in silico models are considered a promising and inevitable tool for the understanding of this complex system. Hence, we need to incorporate the information of the differentiation dependent protein expression in cell based systems biological models of tissue homeostasis. Such methods require measuring tissue differentiation quantitatively while correlating it with biomarker expression intensities.ResultsDifferentiation of a keratinocyte is characterized by its continuously changing morphology concomitant with its movement from the basal layer to the surface, leading to a decreased average nuclei density throughout the tissue. Based thereon, we designed and evaluated three different mathematical measures (nuclei based, distance based, and joint approach) for quantifying differentiation in epidermal keratinocytes. We integrated them with an immunofluorescent staining and image analysis method for tissue sections, automatically quantifying epidermal differentiation and measuring the corresponding expression of biomarkers. When studying five well-known differentiation related biomarkers in an epidermal neck sample only the resulting biomarker profiles incorporating the relative distance information of cells to the tissue borders (distance based and joint approach) provided a high-resolution view on the whole process of keratinocyte differentiation. By contrast, the inverse nuclei density approach led to an increased resolution at early but heavily decreased resolution at late differentiation. This effect results from the heavy non-linear decay of DAPI intensity per area, probably caused by cytoplasmic growth and chromatin decondensation. In the joint approach this effect could be compensated again by incorporating distance information.ConclusionWe suppose that key mechanisms regulating tissue homeostasis probably depend more on distance information rather than on nuclei reorganization. Concluding, the distance approach appears well suited for comprehensively observing keratinocyte differentiation.

Molecules Contributing to the Maintenance of Periodontal Tissues

This review is aimed at providing a depiction of molecules and their topography which characterize native gingiva and PDL fibroblasts, to describe their function in tissue maintenance, and to discuss their possible modulation due to orthodontic tooth movement.Maintenance of the human periodontium requires the balance of proliferation and differentiation in the respective tissues’ cells. Moreover, the cells must synthesize the extracellular matrix molecules and receptors that facilitate adhesion. To describe the molecules that contribute to periodontal tissue maintenance, we illustrate the localization of their expression and topography on frozen sections from native gingival tissue and primary cell cultures derived from periodontal ligament. In native gingival epithelium, proliferation is confined to basal and parabasal cells. Keratin K14, when used as structural marker, is visible in the entire epithelium, while K13, an indicator of early differentiation, is restricted to the suprabasal cell compartment. Vimentin indicates mesenchymal cells in the subgingival connective tissue. Concerning the matrix molecules, collagen type-IV is abundant at the epithelium-lamina propria interface, and fibronectin is apparent throughout the mesenchyme. The matrix receptor integrin β1 reveals a pericellular localization in basal and parabasal cells, while focal adhesion kinase p125FAK is seen pericellularly in all epithelial layers. Cultures of primary periodontal ligament (PDL) fibroblasts (PDL-F) reveal expression of vimentin, strong proliferation, synthesis and extracellular deposition of collagen type-I and fibronectin. The integrin subunits β1 and p125FAK are largely detectable at the cell periphery.ZusammenfassungDieser Übersichtsartikel verfolgt das Ziel, die Moleküle sowie deren Topographie darzustellen, die die native Gingiva und PDL-Fibroblasten charakterisieren, ihre Funktion in der Aufrechterhaltung des Gewebes zu beschreiben und ihre mögliche Veränderung durch orthodontische Zahnbewegungen zu diskutieren.Die Aufrechterhaltung des humanen Parodonts erfordert das Gleichgewicht von Proliferation und Differenzierung in den Zellen der entsprechenden Gewebe. Darüber hinaus müssen die Zellen extrazelluläre Matrix-Moleküle und Rezeptoren synthetisieren, die die Zelladhäsion ermöglichen. Um Moleküle zu beschreiben, die an der Aufrechterhaltung des humanen Parodonts beteiligt sind, stellen wir ihre Lokalisation und Topographie an Gefrierschnitten von nativem Gingiva-Gewebe und Primärkulturen von Zellen des Parodontalligaments dar. Im nativen Gingiva-Epithel ist die Proliferation auf die basalen und parabasalen Zellen beschränkt. Keratin K14 ist überall im Epithel sichtbar, wenn es als Strukturmarker verwendet wird, während K13, ein Marker für die frühe Zelldifferenzierung, auf das suprabasale Zellkompartiment beschränkt bleibt. Vimentin kennzeichnet die mesenchymalen Zellen im subgingivalen Bindegewebe. In Bezug auf die Matrix-Moleküle konzentriert sich Kollagen Typ-IV auf den Übergangsbereich von Epithel und Lamina propria, Fibronektin ist im gesamten Mesenchym vorhanden. Der Matrix-Rezeptor Integrin β1 zeigt eine perizelluläre Lokalisation in basalen sowie parabasalen Zellen, während die fokale Adhäsionskinase p125FAK perizellulär in allen Epithelschichten nachweisbar ist. Primärkulturen von Parodontalligament-(PDL)-Fibroblasten (PDL-F) zeigen eine Expression von Vimentin, eine starke Proliferation und die Synthese sowie extrazelluläre Deposition von Kollagen Typ-I und Fibronektin. Die Integrin-Untereinheit β1 und p125FAK sind größtenteils an der Zellperipherie nachweisbar.

Elevation of collagen type I in fibroblast–keratinocyte cocultures emphasizes the decisive role of fibroblasts in the manifestation of the phenotype of cyclosporin A-induced gingival overgrowth

BACKGROUND AND OBJECTIVEnCollagen type I elevation in cyclosporin A-induced gingival overgrowth supports evidence that gingival fibroblasts play a decisive role in the manifestation of the phenotype. To analyze the role of gingival fibroblasts under more in vivo-like conditions, we evaluated the effect of cyclosporin A on collagen type I gene and protein expression in gingival overgrowth-derived gingival fibroblasts established as cocultures with gingival keratinocytes as well as in matched gingival fibroblast monolayers.nnnMATERIAL AND METHODSnMonolayers and cocultures of primary gingival fibroblasts were treated with cyclosporin A for 6 and 72 h. The expression of collagen type I mRNA was analyzed by quantitative real time polymerase chain reaction, while expression and secretion of collagen type I protein was analyzed by indirect immunofluorescence and western blotting.nnnRESULTSnCompared with controls, significant elevation of collagen type I mRNA was restricted to cocultures after 6 and 72 h of treatment with cyclosporin A. In keratinocytes, collagen type I remained undetectable. In monolayers and cocultures, indirect immunofluorescence showed a slightly higher level of collagen type I protein in gingival fibroblasts in response to stimulation with cyclosporin A. Semiquantitative detection of collagen type I by western blotting demonstrated a nonsignificant increase for cell extracts in monolayers and cocultures. For secreted collagen type I, western blot analysis of the supernatants revealed elevated protein levels in cultures stimulated with cyclosporin A. Compared with the corresponding monolayers, the stimulatory effect of cyclosporin A on protein secretion was significant only in coculture.nnnCONCLUSIONnOur results indicate that collagen type I is a target of cyclosporin A and that gingival fibroblasts are decisive for the manifestation of the gingival overgrowth-phenotype. Furthermore, the results suggest that cocultures of gingival overgrowth-derived gingival fibroblasts and gingival keratinocytes permit analysis of cyclosporin A-induced effects under more in vivo-like conditions.

Discrimination of epithelium-like and fibroblast-like phenotypes derived from ethanol-treated immortalised human gingival keratinocytes in epithelial equivalents

Ethanol treatment of immortalised human gingival keratinocytes (IHGK) yields in an epithelium-like (EPI) and fibroblast-like (FIB) phenotype. With respect to the stratified gingival epithelium, putative structural and molecular differences assigning cells to these phenotypes have not, to date, been analysed in a three-dimensional tissue/epithelial context. Therefore, we generated epithelial equivalents (EEs) in organotypic co-cultures of IHGK, EPI and FIB cells for 1 and 2xa0weeks and conducted protein and gene expression studies on the EEs for epithelial biomarkers including keratin K14, integrin subunits α6 and β1, E-cadherin, and mesenchymal vimentin. As in the EEs of IHGK and EPI, indirect immunofluorescence revealed continuous expression of β1 integrin in EEs of FIB cells. However, FIB cells exhibited a significant down-regulation in K14 and integrin α6 protein and a loss of E-cadherin at week 2, whereas vimentin was increased. FIB EEs were devoid of transcripts for E-cadherin at both time points, although transcription of the other genes remained constant in all phenotypes. Thus, the FIB phenotype exhibited a poor epithelial structure coinciding with disturbances in the expression of epithelial biomarkers and the persistence of mesenchymal vimentin. Transcription analysis revealed post-transcriptional regulation of vimentin in IHGK and EPI and of K14 and α6 in FIB cells. Our findings indicate that differences in the epithelial integrity and expression of molecules in EEs allow for the discrimination of EPI and FIB cells. This suggests that FIB cells share features of epithelial-mesenchymal transition and reflect a more progressive stage in epithelial cell transformation.

Spatial quantification and classification of skin response following perturbation using organotypic skin cultures

MOTIVATIONnFor a mechanistic understanding of skin and its response to an induced perturbation, systems biology is gaining increasing attention. Unfortunately, quantitative and spatial expression data for skin, like for most other tissues, are almost not available.nnnRESULTSnIntegrating organotypic skin cultures, whole-slide scanning and subsequent image processing provides bioinformatics with a novel source of spatial expression data. We here used this approach to quantitatively describe the effect of treating organotypic skin cultures with sodium dodecyl sulphate in a non-corrosive concentration. We first measured the differentiation-related spatial expression gradient of Heat-Shock-Protein 27 in a time series of up to 24 h. Secondly, a multi-dimensional tissue classifier for predicting skin irritation was developed based on abstract features of these profiles. We obtained a high specificity of 0.94 and a sensitivity of 0.92 compared with manual classification. Our results demonstrate that the integration of tissue cultures, whole-slide scanning and image processing is well suited for both the standardized data acquisition for systems biological tissue models and a highly robust classification of tissue responses.