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Kritzenberger M, Junglas B, Framme C, Helbig H, Gabel V-P, Fuchshofer R, Tamm E R & Hillenkamp J (2011) Histopathology , 953-965 To identify differences in extracellular matrix contents between idiopathic epiretinal membranes (IEM) of cellophane macular reflex (CMRM) or preretinal macular fibrosis (PMFM) type. Idiopathic epiretinal membranes were analysed by light and quantitative transmission electron microscopy, immunohistochemistry and Western blotting. Substantial differences between CMRM and PMFM were observed regarding the nature of extracellular fibrils. In CMRM the fibrils were thin, with diameters between 6 and 15 nm. Between the fibrils, aggregates of long-spacing collagen were observed. In PMFM the diameters of fibrils measured either 18-26 or 36-56 nm. Using immunogold electron microscopy, 6-15 nm fibrils in CMRM were labelled for collagen type VI, while the fibrils in PMFM remained unstained. Using Western blotting and immunohistochemistry, a strong signal for collagen type VI was observed in all CMRM, while immunoreactivity was weak or absent in PMFM. In contrast, PMFM showed immunoreactivity for collagen types I and II, which was weak or absent in CMRM. Both types of membranes showed immunoreactivity for collagen types III and IV, laminin and fibronectin with similar intensity. The presence of high amounts of collagen type VI in CMRM and the relative absence of collagen types I and II is the major structural difference to PMFM. [ABSTRACT FROM AUTHOR]

Mutations in myocilin, a 55–57 kDa secreted glycoprotein, are causative for some forms of primary open-angle glaucoma (POAG). In vitro studies indicate that myocilin can modulate the hydrodynamic outflow resistance in the trabecular meshwork (TM) and that elevated amounts of myocilin can obstruct the TM outflow system in POAG. In this study, we analyzed the localization of myocilin in the trabecular meshwork (TM) of eyes with primary open-angle glaucoma (POAG), and compared it with that of normal eyes. Immunohistochemistry for myocilin was performed in the eyes of human donors (nine normal and 14 with POAG, including one with steroid-induced glaucoma). Staining for myocilin was observed in the extracellular spaces of the juxtacanalicular tissue (JCT) in all normal eyes. Some normal eyes did also show cytoplasmic staining for myocilin in TM cells. In the eyes of six donors with POAG, staining of the JCT was more widespread and intense than in normal eyes. In the other eyes with POAG, immunoreactivity for myocilin in the JCT was not markedly different to that of normal eyes. Staining intensity in the JCT of POAG eyes did not obviously correlate with intraocular pressure or clinical severity. In the eyes of one patient with steroid-induced POAG, cells of the TM, Schlemm’s canal endothelium, and the anterior stroma of the iris showed an immunoreactivity for myocilin which was considerably more intense than in normal eyes, or in the eyes with other forms of POAG. In some cases of POAG, the structural changes in the JCT include an increase in myocilin in the extracellular pathways of aqueous humor. Treatment with steroids appears to increase myocilin synthesis in TM and iris of human eyes in situ. [ABSTRACT FROM AUTHOR]

Abstract: A distinct structural change in the trabecular meshwork (TM) of patients with primary open-angle glaucoma (POAG) is the increase in fibrillar extracellular matrix (ECM) in the juxtacanalicular region of the TM. Transforming growth factor (TGF)-β2 signaling may be involved, as TGF-β2 is significantly increased in the aqueous humor of patients with POAG. In cultured human TM cells, TGF-β2 causes an increase in ECM deposition, an effect that is blunted or prevented, if BMP7 is added in combination with TGF-β2. In order to know more about the signaling network that is induced in HTM cells treated with BMP7, TGF-β2 or the combination of both factors, we identified differentially regulated genes by microarray analysis, and confirmed selected genes by quantitative RT-PCR, Western blotting, or immunohistochemistry. We observed multiple effects of both TGF-β2 and BMP7 on the expression of a considerable number of genes involved in growth factor signaling, ECM structure and turnover, and modification of the cytoskeleton. Among the genes that were found to be regulated were CAPZA1, CDC42BPB, EFEMP1, FGF5, FSTL3, HBEGF, LTBP1, LTBP2, MATN2, NRP1, SERPINE1, SH3MD1, SMTN, SMAD7, TFPI2, TNFAIP6, and VEGF. Since SMAD7 encodes for Smad7, an inhibitory Smad that acts in a negative-feedback loop to inhibit TGF-β activity, we silenced Smad7 mRNA in cultured human TM cells by a specific small interfering RNA. Silencing of its mRNA caused a substantial knock down of Smad7 in TM cells. Following combined BMP7/TGF-β2 treatment, the antagonizing effect of BMP7 on TGF-β2-induced CTGF expression was abolished. We conclude that Smad7 is the key molecular switch that inhibits TGF-β2 signaling, and mediates the blunting effects of BMP7 on TGF-β2 in TM cells. A therapeutic modulation of Smad7 might be a promising approach to influence ECM turnover in the TM and to treat POAG. [Copyright &y& Elsevier]

Abstract: Mutations in the OPTN gene encoding for optineurin have been associated with primary open-angle glaucoma. The functional role(s) of optineurin in the normal and glaucomatous eye are unclear. As optineurin interferes with TNF-α mediated cell death in vitro, an involvement of optineurin in the regulatory pathways leading to apoptosis of retinal ganglion cells has been suggested. The goal of the present study was to study the molecular properties of optineurin and its capabilities to prevent apoptosis in vivo in the eyes of transgenic mice. The chicken βB1-crystallin promoter was used to overexpress ectopic optineurin in the lenses of transgenic mice. The expression of transgenic mRNA was monitored by northern blot analysis. The localization of transgenic optineurin was investigated by one- and two-dimensional western blot analysis and by immunohistochemistry, and compared with that of endogenous optineurin. To assess effects of transgenic optineurin on apoptosis, βB1-crystallin-OPTN mice were crossbred with βB1-crystallin-TGFβ1 mice that undergo substantial TGF-β1-induced apoptotic cell death in the lens. Two independent βB1-crystallin-OPTN transgenic lines were established, in which transgenic optineurin was expressed strictly lens-specific as assessed by Northern and Western blotting, and by immunohistochemistry. In contrast, endogenous optineurin was preferentially expressed in the retina, where retinal ganglion cells showed strong labeling. Immunostaining for endogenous optineurin in the anterior eye was considerably weaker than in the posterior eye and was seen in iris, ciliary epithelium, cells of corneal stroma and endothelium, and in the trabecular meshwork. Neither transgenic nor endogenous optineurin was found in the aqueous humor. Transgenic overexpression of optineurin did not have measurable effects on TGFβ1-induced apoptosis in mixed βB1-crystallin-OPTN/βB1-crystallin-TGFβ1 transgenic mice. Our results show that optineurin is a cytoplasmatic rather than a secretory protein that is preferentially expressed in retinal ganglion cells, and argue against a major role of optineurin for the modulation of apoptosis in vivo. [Copyright &y& Elsevier]

Myocilin is expressed in the glomerulus of the kidney and induced in mesangioproliferative glomerulonephritis.Background.Myocilin is a 55 to 57 kD secreted glycoprotein and member of the olfactomedin protein family. It is expressed in high amounts in the outflow tissues of the aqueous humor in the eye where it is supposed to contribute to outflow resistance. Myocilin is mutated in some forms of primary open angle glaucoma and affected patients show very high intraocular pressures because of an increase in resistance to aqueous humor outflow. To obtain information, if myocilin may play a comparable role in other tissues with transendothelial fluid flow, we investigated its expression in the rat kidney.Methods.The expression of myocilin in the normal rat kidney and its changes during mesangioproliferative glomerulonephritis were investigated by immunohistochemistry, one- and two-dimensional gel electrophoresis with Western blotting, and reverse transcription-polymerase chain reaction (RT-PCR).Results.Myocilin and its mRNA were detected in isolated glomeruli. Immunohistochemistry showed specific labeling of glomerular cells, while tubular and interstitial regions were essentially negative. Double staining with the podocyte-specific markers synaptopodin and ezrin indicated that myocilin-positive cells were predominately podocytes. During mesangioproliferative glomerulonephritis, an induction of myocilin immunoreactivity was observed. Labeling for myocilin was now observed in activated mesangial cells and areas of glomerular sclerosis. In parallel cell culture experiments, mRNA for myocilin was detected in cultured murine podocytes and rat mesangial cells.Conclusion.Myocilin is expressed in podocytes of the kidney and induced in mesangial cells during experimental mesangioproliferative glomerulonephritis. The specific function of myocilin in the kidney is not clear, but in a parallel to functions of other olfactomedin proteins, it might have a role in cell-cell adhesion and/or signaling processes. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β2 (TGF-β2) is elevated in the aqueous humor of patients with primary open-angle glaucoma (POAG), and high levels of TGF-β2 are thought to contribute to the pathogenesis of POAG. Most TGF-β2 in the eye is present in a latent, inactive form and the mechanisms of its in vivo activation are unclear. Since thrombospondin-1 (TSP-1) is one of the most potent in vivo activating molecules of TGF-βs, we investigated the localization and expression of TSP-1 in the aqueous humor outflow pathways. TSP-1 immunohistochemistry was performed in the eyes of human donors (8 normal and 17 with glaucoma). In addition, the eyes of Tsp-1-/--deficient mice and normal Tsp-1+/+ mice were investigated. TSP-1 mRNA expression was assessed by reverse transcription-polymerase chain reaction and Northern blotting of RNA from fresh trabecular meshwork (TM), and human and mouse TM cells in vitro. In addition, Northern and Western blot analyses of TM cells after incubation with TGF-β and dexamethasone were performed. In most of the eyes, TSP-1 immunolabeling was predominately observed in extracellular areas of the juxtacanalicular (cribriform) part of the TM. Some focal staining was observed in the corneoscleral and uveal parts of the TM. In the eyes of six glaucoma patients (including one with steroid-induced glaucoma), TSP-1 immunoreactivity was considerably more intense and all regions of the TM were positively labeled. In double labeling experiments, staining for TSP-1 did not overlap with that of fibronectin or type VI collagen. mRNA for TSP-1 was detected in both fresh and cultured TM cells. Incubation of TM cells with TGF-β1 and dexamethasone caused a marked increase in TSP-1 expression. TSP-1 in the TM might act as a potent local endogenous activator of TGF-βs in the aqueous humor and mediate any local effects of TGF-β and/or dexamethasone on the outflow of aqueous humor. [Copyright &y& Elsevier]