Regulation of Connective Tissue Growth Factor Expression Influences the Cell Viability In Human Trabecular Meshwork Cells

Connective tissue growth factor (CTGF) induces extracellular matrix (ECM) synthesis and contractility in human trabecular meshwork (HTM) cells. Both processes are involved in the pathogenesis of primary open-angle glaucoma. To date, little is known about regulation and function of CTGF expression in the trabecular meshwork (TM). Therefore, we analysed the effects of different aqueous humour proteins and stressors on CTGF expression in HTM cells. HTM cells from three different donors were treated with endothelin-1, insulin-like growth factor (IGF)-1, angiotensin-II, H2O2 and heat shock and were analysed by immunohistochemistry, real-time RT-PCR and Western blotting. Viability after H2O2 treatment was measured in CTGF silenced HTM-N cells and their controls. Latrunculin A reduced expression of CTGF by about 50% compared to untreated HTM cells, whereas endothelin-1, IGF-1, angiotensin-II, heat shock and oxidative stress led to a significant increase. Silencing of CTGF resulted in a delayed expression of αB-crystallin and in reduced cell viability in comparison to the controls after oxidative stress. Conversely, CTGF treatment led to a higher cell viability rate after H2O2 treatment. CTGF expression is induced by factors that have been linked to glaucoma. An increased level of CTGF appears to protect TM cells against damage induced by stress. The beneficial effect of CTGF for viability of TM cells is likely associated with the effects on increased ECM synthesis and higher contractility of the TM, thereby contributing to reduced aqueous humour outflow facility causing increased intraocular pressure

Ignicoccus hospitalis and Nanoarchaeum equitans: ultrastructure, cell–cell interaction, and 3D reconstruction from serial sections of freeze-substituted cells and by electron cryotomography

Ultrastructure and intercellular interaction of Ignicoccus hospitalis and Nanoarchaeum equitans were investigated using two different electron microscopy approaches, by three-dimensional reconstructions from serial sections, and by electron cryotomography. Serial sections were assembled into 3D reconstructions, for visualizing the unusual complexity of I. hospitalis, its huge periplasmic space, the vesiculating cytoplasmic membrane, and the outer membrane. The cytoplasm contains fibres which are reminiscent to a cytoskeleton. Cell division in I. hospitalis is complex, and different to that in Euryarchaeota or Bacteria. An irregular invagination of the cytoplasmic membrane is followed by separation of the two cytoplasms. Simultaneous constriction of cytoplasmic plus outer membrane is not observed. Cells of N. equitans show a classical mode of cell division, by constriction in the mid-plane. Their cytoplasm exhibits two types of fibres, elongated and ring-shaped. Electron micrographs of contact sites between I. hospitalis and N. equitans exhibit two modes of interaction. One is indirect and mediated by thin fibres; in other cells the two cell surfaces are in direct contact. The two membranes of I. hospitalis cells are frequently seen in direct contact, possibly a prerequisite for transporting metabolites or substrates from the cytoplasm of one cell to the other. Rarely, a transport based on cargo vesicles is observed between I. hospitalis and N. equitans

Untersuchungen zur molekularen Funktion des Connective Tissue Growth Factor im Trabekelwerk

Der Connective Tissue Growth Factor (CTGF) ist ein sezerniertes Glykoprotein aus der Familie der CCN-Proteine. CTGF ist aufgrund seiner Multidomänenstruktur in der Lage an verschiedenste Transmembran-, extrazelluläre Matrix (EZM)- sowie Signalproteine zu binden und deren Funktion zu beeinflussen. So stimuliert oder vermittelt CTGF die Adhäsion, Migration und Proliferation von Zellen und ist als Wachstumsfaktor essentiell für die Entwicklung bestimmter Gewebe. Besondere Beachtung findet CTGF allerdings im Zusammenhang mit der Pathogenese fibrotischer Erkrankungen. In diesen Fällen wird CTGF oftmals als Downstream-Mediator des pro-fibrotischen Faktors Transforming Growth Factor (TGF)-β gehandelt. TGF-β2 wird mit der Pathogenese des primären Offenwinkelgalukoms (POWG) in Zusammenhang gebracht, was auch eine Rolle von CTGF bei der Pathogenese dieser Krankheit nahelegt. Ziel der vorliegenden Arbeit war es die Funktionen von CTGF im Trabekelwerk des Auges zu charakterisieren. Dazu sollten zunächst Signalmoleküle identifiziert werden, die CTGF im Trabekelwerk induzieren. Anschließend sollte CTGF in einem eukaryotischen System rekombinant hergestellt und aufgereinigt werden, um über in vitro Versuche den Einfluss von CTGF auf humane Trabekelwerkzellen zu analysieren. Diese Ergebnisse sollten in vivo über die Entwicklung von transgenen Mäusen mit einer CTGF-Überexpression überprüft werden. Um die physiologische Funktion von CTGF in humanen Trabekelwerkzellen zu untersuchen, sollte ein stabiler Knockdown von CTGF durchgeführt werden. Neben TGF-β waren die im KW vorhandenen Faktoren Endothelin-1, Angiotensin II und IGF-1 in der Lage die Expression von CTGF in humanen TWZ zu erhöhen. Rekombinantes CTGF konnte nach Anfügen eines His-Tags an den C-Terminus durch Ausnutzung seiner Affinität zu Heparin sowie zu Ni2+-NTA aufgereinigt werden. Die biologische Funktionalität konnte nach Behandlung von TWZ bestätigt werden. CTGF induzierte die Expressionen wichtiger EZM-Proteine wie FN und mehrerer Kollagene, sowie die der Integrin-Untereinheiten αV und β1. und vermittelte die Effekte von TGF-β auf die Expression dieser Proteine. Die Effekte von TGF-β auf das extrazelluläre proteolytische Systeme zeigten sich nicht durch CTGF vermittelt. Neben den Veränderungen an EZM und Integrinen konnten auch Effekte auf das Aktinzytoskelett beobachtet werden. CTGF erhöhte zum einen die Expression bedeutender Komponenten wie etwa α-smAktin. Zum anderen führte die Behandlung zur Beeinflussung von intrazellulären Signalmolekülen, die für die Regulation des Aktinzytoskeletts verantwortlich sind. CTGF konnte die Aktivität der kleinen GTPase RhoA, der Myosin Light Chain, der Focal Adhesion Kinase sowie der Extracellular Signal Regulated Kinase erhöhen. Dies führte unter anderem zu einer Zunahme an Aktin-Stressfasern in den Zellen und zu einer erhöhten Kontraktilität. In einem in vivo-Mausmodell mit einer Augen-spezifischen CTGF-Überexpression konnte ebenso eine Vermehrung von FN und α-smAktin im TW nachgewiesen werden. Zudem wiesen die transgenen Mäuse einen Anstieg des IOD auf. Ein stabiler Knockdown von CTGF konnte in immortalisierten TWZ mit Hilfe eines pSilencer-Vektors erreicht werden. Dies führte zu einer verminderten Expression einiger Effektorproteine von CTGF und zu einer reduzierten oder vollständig blockierten Aktivität derjenigen Signalmoleküle, die in Wildtypzellen durch CTGF aktiviert wurden. In zellphysiologischen Untersuchungen konnte weiterhin eine reduzierte migratorische und proliferative Aktivität aufgezeigt werden. Die Adhäsion an Oberflächen hingegen wurde durch den CTGF-Knockdown verstärkt. Eine Behandlung mit rekombinantem CTGF konnte im Falle der Proliferation sowie der Adhäsion eine Angleichung an den Wildtyp herstellen, führte aber zu einem weiteren Absinken der Expression von CTGF Effektorproteinen. Zusammenfassend zeigen die Ergebnisse dieser Arbeit, dass CTGF nicht nur in der Lage ist, die EZM sondern auch die Kontraktilität des TW zu beeinflussen. Diese Veränderungen können zu einem Anstieg des intraokulären Drucks und damit zu einem erhöhten Risiko für die Erkrankung des POWG führen. Somit kann CTGF als ein kritischer Faktor in der Pathogenese des POWG angesehen werden

The interaction of Nanoarchaeum equitans with Ignicoccus hospitalis: proteins in the contact site between two cells

The two archaea Ignicoccus hospitalis and Nanoarchaeum equitans form a unique intimate association, the character of which is not yet fully understood. Electron microscopic investigations show that at least two modes of cell-cell interactions exist: (i) the two cells are interconnected via thin fibres; and (ii) the two cell surfaces are in direct contact with each other. In order to shed further light on the molecules involved, we isolated a protein complex, by using detergent-induced solubilization of cell envelopes, followed by a combination of chromatography steps. Analysis by MS and comparison with databases revealed that this fraction contained two dominant proteins, representing the respective major envelope proteins of the two archaea. In addition, a considerable set of membrane proteins is specifically associated with these proteins. They are now the focus of further biochemical and ultrastructural investigations

Nanoarchaeum equitans and Ignicoccus hospitalis: New Insights into a Unique, Intimate Association of Two Archaea▿

Nanoarchaeum equitans and Ignicoccus hospitalis represent a unique, intimate association of two archaea. Both form a stable coculture which is mandatory for N. equitans but not for the host I. hospitalis. Here, we investigated interactions and mutual influence between these microorganisms. Fermentation studies revealed that during exponential growth only about 25% of I. hospitalis cells are occupied by N. equitans cells (one to three cells). The latter strongly proliferate in the stationary phase of I. hospitalis, until 80 to 90% of the I. hospitalis cells carry around 10 N. equitans cells. Furthermore, the expulsion of H2S, the major metabolic end product of I. hospitalis, by strong gas stripping yields huge amounts of free N. equitans cells. N. equitans had no influence on the doubling times, final cell concentrations, and growth temperature, pH, or salt concentration ranges or optima of I. hospitalis. However, isolation studies using optical tweezers revealed that infection with N. equitans inhibited the proliferation of individual I. hospitalis cells. This inhibition might be caused by deprivation of the host of cell components like amino acids, as demonstrated by 13C-labeling studies. The strong dependence of N. equitans on I. hospitalis was affirmed by live-dead staining and electron microscopic analyses, which indicated a tight physiological and structural connection between the two microorganisms. No alternative hosts, including other Ignicoccus species, were accepted by N. equitans. In summary, the data show a highly specialized association of N. equitans and I. hospitalis which so far cannot be assigned to a classical symbiosis, commensalism, or parasitism

Different collagen types define two types of idiopathic epiretinal membranes

To identify differences in extracellular matrix contents between idiopathic epiretinal membranes (IEM) of cellophane macular reflex (CMRM) or preretinal macular fibrosis (PMFM) type

The dominating outer membrane protein of the hyperthermophilic Archaeum Ignicoccus hospitalis: a novel pore-forming complex

The membrane protein Imp1227 (Ignicoccus outer membrane protein; Imp1227) is the main protein constituent of the unique outer sheath of the hyperthermophilic, chemolithoautotrophic Archaeum Ignicoccus hospitalis. This outer sheath is the so far only known example for an asymmetric bilayer among the Archaea and is named 'outer membrane'. With its molecular mass of only 6.23 kDa, Imp1227 is found to be incorporated into the outer membrane in form of large, stable complexes. When separated by SDS-PAGE, they exhibit apparent masses of about 150, 50, 45 and 35 kDa. Dissociation into the monomeric form is achieved by treatment with SDS-containing solutions at temperatures at or above 113 degrees C. Electron micrographs of negatively stained samples confirm that isolated membranes are tightly packed with round complexes, about 7 nm in diameter, with a central, stain-filled 2 nm pore; a local two-dimensional crystalline arrangement in form of small patches can be detected by tomographic reconstruction. The comparison of the nucleotide and amino acid sequence of Imp1227 with public databases showed no reliable similarities with known proteins. Using secondary structure prediction and molecular modelling, an alpha-helical transmembrane domain is proposed; for the oligomer, a ring-shaped nonamer with a central 2 nm pore is a likely arrangement