Benjamin Junglas

Connective Tissue Growth Factor Causes Glaucoma by Modifying the Actin Cytoskeleton of the Trabecular Meshwork

The most critical risk factor for optic nerve damage in cases of primary open-angle glaucoma (POAG) is an increased intraocular pressure (IOP) caused by a resistance to aqueous humor outflow in the trabecular meshwork (TM). The molecular pathogenesis of this increase in outflow resistance in POAG has not yet been identified, but it may involve transforming growth factor TGF-β2, which is found in higher amounts in the aqueous humor of patients with POAG. Connective tissue growth factor (CTGF) is a TGF-β2 target gene with high constitutive TM expression. In this study, we show that either adenoviral-mediated or transgenic CTGF overexpression in the mouse eye increases IOP and leads to optic nerve damage. CTGF induces TM fibronectin and α-SMA in animals, whereas actin stress fibers and contractility are both induced in cultured TM cells. Depletion of CTGF by RNA interference leads to a marked attenuation of the actin cytoskeleton. Rho kinase inhibitors cause a reversible decline in the IOP of CTGF-overexpressing mice to levels seen in control littermates. Overall, the effects of CTGF on IOP appear to be caused by a modification of the TM actin cytoskeleton. CTGF-overexpressing mice provide a model that mimics the essential functional and structural aspects of POAG and offer a molecular mechanism to explain the increase of its most critical risk factor.

Connective tissue growth factor induces extracellular matrix deposition in human trabecular meshwork cells

The major structural change in the human trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG) is an increase in extracellular matrix (ECM) in the juxtacanalicular region of the TM. There is evidence that treatment with TGF-beta2 causes an induction of ECM deposition in cultured human TM cells and that TGF-beta2 is causatively involved in the JCT ECM increase in POAG. In the present study, we investigated the effects of connective tissue growth factor (CTGF) on the biology of cultured human TM cells. CTGF is a downstream mediator of TGF-beta2-signaling, which is expressed at high amounts in the human TM in situ. HEK293 cells were transfected with an eukaryotic expression plasmid containing the coding sequences of human CTGF. Secreted CTGF was isolated and purified by chromatography. Primary human TM cells were incubated for 24 h with CTGF at concentrations of 2.5-100 ng/ml. Following treatment with CTGF, the expression of various ECM components that are expressed in the JCT, matrix metalloproteinases (MMPs) and integrins was investigated by real-time RT-PCR and western blot analyses. In addition, the activity of MMPs was investigated by gelatine zymography. The effect of CTGF silencing on TGF-beta2-induced gene expression was investigated by transfection of immortalized HTM cells with CTGF-specific small interfering (si)RNA before TGF-beta2 treatment. CTGF-treated human TM cells showed an increase in the expression of fibronectin, collagen types I, III, IV and VI, as well as in the integrin subunits aV and beta1. Lower concentrations of CTGF caused an autoinduction of CTGF expression. No effects were observed on the expression and activity of MMP-2, MMP-9 and plasminogen activator inhibitor-1 (PAI-1). Transfection with CTGF-specific siRNA inhibited the TGF-beta2-induced upregulation of CTGF and fibronectin. Our results indicate that treatment of human TM cells with recombinant CTGF causes distinct changes in gene expression and that CTGF is a critical mediator of the effects of TGF-beta2 on ECM synthesis in human TM cells. An intriguing aspect supported by the data of the present work is that the pharmacologic modulation of CTGF might be a useful approach to develop novel therapeutic strategies to prevent or to reverse the structural changes that occur in the TM of eyes with POAG.

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.

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.

CTGF is overexpressed in malignant melanoma and promotes cell invasion and migration

Background:Malignant melanoma cells are known to have altered expression of growth factors compared with normal human melanocytes. These changes most likely favour tumour growth and progression, and influence tumour environment. The induction of transforming growth factor beta1, 2 and 3 as well as BMP4 and BMP7 expression in malignant melanoma has been reported before, whereas the expression of an important modulator of these molecules, connective tissue growth factor (CTGF), has not been investigated in melanomas until now.Methods:Expression of CTGF was analysed in melanoma cell lines and tissue samples by qRT–PCR and immunohistochemistry. To determine the regulation of CTGF expression in malignant melanoma, specific siRNA was used. Additionally, migration, invasion and attachment assays were carried out.Results:We were able to demonstrate that CTGF expression is upregulated in nine melanoma cell lines and in primary and metastatic melanoma in situ. The transcription factor HIF-1α was revealed as a positive regulator for CTGF expression. Melanoma cells, in which CTGF expression is diminished, show a strong reduction of migratory and invasive properties when compared with controls. Further, treatment of normal human epidermal melanocytes with recombinant CTGF leads to an increase of migratory and invasive behaviour of these cells.Conclusion:These results suggest that CTGF promotes melanoma cell invasion and migration and, therefore, has an important role in the progression of malignant melanoma.

Different collagen types define two types of idiopathic epiretinal membranes

Kritzenberger M, Junglas B, Framme C, Helbig H, Gabel V‐P, Fuchshofer R, Tamm E R & Hillenkamp J
(2011) Histopathology 58, 953–965

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°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 α‐helical transmembrane domain is proposed; for the oligomer, a ring‐shaped nonamer with a central 2 nm pore is a likely arrangement.

The role of plasmalemma vesicle-associated protein (PLVAP) in endothelial cells of Schlemm's canal and ocular capillaries.

Plasmalemma vesicle-associated protein (PLVAP, PV-1) is an endothelial protein that specifically localizes to diaphragms of fenestrae in fenestrated capillaries, and to stomatal diaphragms of caveolae. Here we investigated the localization of PLVAP in Schlemms canal endothelium and ocular capillaries, and studied the structural effects of PLVAP deficiency. In mouse, pig and human eyes, immunoreactivity for PLVAP was present in fenestrated capillaries of choroid and ciliary processes, but not in the continuous capillaries of retina and ciliary muscle. In all three species staining for PLVAP was seen in the endothelia of the outflow vessels of aqueous humor e.g. Schlemms canal (SC, mouse and human), aqueous plexus (AP, pig) and the scleral collector channels. Essentially comparable findings were observed when the expression of β-galactosidase was investigated in mutant heterozygous and homozygous PLVAP-deficient mice with LacZ inserted into the Plvap locus. By transmission electron microscopy, the vast majority of caveolae in SC endothelial cells showed a stomatal diaphragm. In addition, solitary fenestrae or minipores with a diaphragm were occasionally observed in SC or AP of all three species. In contrast, mutant Plvap(-/-) mice showed a complete absence of stomatal diaphragms in SC caveolae while no SC minipores were observed. Moreover, diaphragms were absent in fenestrae of endothelial cells in the capillaries of the ciliary processes or the choriocapillaris, findings which were associated with a substantial decrease in the number of fenestrae. PLVAP is expressed in endothelial cells of Schlemms canal and is essential for the formation of diaphragms in vascular endothelial cells of the eye.

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.

The regulation of connective tissue growth factor expression influences the viability of 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.