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Abstract: Intraocular pressure (IOP) is the most critical risk factor for primary open angle glaucoma (POAG). In most cases of POAG, IOP is increased because of an abnormally high aqueous humor outflow resistance in the juxtacanalicular region of the trabecular meshwork. A distinct structural change in the trabecular meshwork of patients with POAG is the increase in fibrillar extracellular matrix in the juxtacanalicular region of the trabecular meshwork. Our knowledge on the molecular factors that govern turnover of the extracellular matrix in the trabecular meshwork has increased considerably in recent years. It has become clear that quality and quantity of the extracellular matrix in the trabecular meshwork are regulated by several signaling molecules that interact with each other to promote its synthesis, degradation, or extracellular modification. Transforming growth factor-β1 and β2 (TGF-β1 and TGF-β2) which derive from the aqueous humor or may be locally expressed induce in cultured trabecular meshwork cells the expression of a variety of extracellular matrix molecules. The action of TGF-βs very likely requires local activation by thrombospondin-1 and is partly mediated by its downstream mediator connective tissue growth factor, both of which are constitutively expressed in the trabecular meshwork. Bone morphogenetic proteins (BMP)-7 and -4 effectively antagonize the effects of TGF-β2 on matrix deposition. The antagonizing effects of BMP-7 are mediated in trabecular meshwork cells through Smad7. Smad7 is a key molecular switch to inhibit TGF-β2 signaling in the trabecular meshwork. [Copyright &y& Elsevier]

Abstract: The major drainage structures for aqueous humor (AH) are the conventional or trabecular outflow pathways, which are comprised of the trabecular meshwork (made up by the uveal and corneoscleral meshworks), the juxtacanalicular connective tissue (JCT), the endothelial lining of Schlemm''s canal (SC), the collecting channels and the aqueous veins. The trabecular meshwork (TM) outflow pathways are critical in providing resistance to AH outflow and in generating intraocular pressure (IOP). Outflow resistance in the TM outflow pathways increases with age and primary open-angle glaucoma. Uveal and corneoscleral meshworks form connective tissue lamellae or beams that are covered by flat TM cells which rest on a basal lamina. TM cells in the JCT are surrounded by fibrillar elements of the extracellular matrix (ECM) to form a loose connective tissue. In contrast to the other parts of the TM, JCT cells and ECM fibrils do not form lamellae, but are arranged more irregularly. SC inner wall endothelial cells form giant vacuoles in response to AH flow, as well as intracellular and paracellular pores. In addition, minipores that are covered with a diaphragm are observed. There is considerable evidence that normal AH outflow resistance resides in the inner wall region of SC, which is formed by the JCT and SC inner wall endothelium. Modulation of TM cell tone by the action of their actomyosin system affects TM outflow resistance. In addition, the architecture of the TM outflow pathways and consequently outflow resistance appear to be modulated by contraction of ciliary muscle and scleral spur cells. The scleral spur contains axons that innervate scleral spur cells or that have the ultrastructural characteristics of mechanosensory nerve endings. [Copyright &y& Elsevier]

A root cause for the development and progression of primary open-angle glaucoma might be the loss of the Schlemm's canal (SC) cell function due to an impaired Angiopoietin-1 (Angpt-1)/Tie2 signaling. Current therapeutic options fail to restore the SC cell function. We propose Angpt-1 mimetic nanoparticles (NPs) that are intended to bind in a multivalent manner to the Tie2 receptor for successful receptor activation. To this end, an Angpt-1 mimetic peptide was coupled to a poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block co-polymer. The modified polymer allowed for the fabrication of Angpt-1 mimetic NPs with a narrow size distribution (polydispersity index < 0.2) and the size of the NPs ranging from about 120 nm (100% ligand density) to about 100 nm (5% ligand density). NP interaction with endothelial cells (HUVECs, EA.hy926) as surrogate for SC cells and fibroblasts as control was investigated by flow cytometry and confocal microscopy. The NP–cell interaction strongly depended on the ligand density and size of NPs. The cellular response to the NPs was investigated by a Ca2+ mobilization assay as well as by a real-time RT-PCR and Western blot analysis of endothelial nitric oxide synthase (eNOS). NPs with a ligand density of 25% opposed VEGF-induced Ca2+ influx in HUVECs significantly which could possibly increase cell relaxation and thus aqueous humor drainage, whereas the expression and synthesis of eNOS was not significantly altered. Therefore, we suggest Angpt-1 mimetic NPs as a first step towards a causative therapy to recover the loss of SC cell function during glaucoma. [ABSTRACT FROM AUTHOR]

• Decorin deficiency leads to elevated intraocular pressure in mice. • TGF-β target genes are enriched in the conventional outflow pathways of decorin-deficient mice. • Decorin attenuates the expression of TGF-β2 in cultured HTM cells. • Decorin levels are reduced in the chamber angle of glaucomatous eyes. Primary open-angle glaucoma, a neurodegenerative disorder characterized by degeneration of optic nerve axons, is a frequent cause of vision loss and blindness worldwide. Several randomized multicenter studies have identified intraocular pressure as the major risk factor for its development, caused by an increased outflow resistance to the aqueous humor within the trabecular meshwork. However, the molecular mechanism for increased outflow resistance in POAG has not been fully established. One of the proposed players is the pro-fibrotic transforming growth factor (TGF)-β2, which is found in higher amounts in the aqueous humor of patients with POAG. In this study we elucidated the role of decorin, a small leucine-rich proteoglycan and known antagonist of TGF-β, in the region of aqueous humor outflow tissue. Utilizing decorin deficient mice, we discovered that decorin modulated TGF-β signaling in the canonical outflow pathways and the lack of decorin in vivo caused an increase in intraocular pressure. Additionally, the Dcn −/− mice showed significant loss of optic nerve axons and morphological changes in the glial lamina, typical features of glaucoma. Moreover, using human trabecular meshwork cells we discovered that soluble decorin attenuated TGF-β2 mediated synthesis and expression of typical downstream target genes including CCN2/CTGF, FN and COL IV. Finally, we found a negative reciprocal regulation of decorin and TGF-β, with a dramatic downregulation of decorin in the canonical outflow pathways of patients with primary open-angle glaucoma. Collectively, our results indicate that decorin plays an important role in the pathogenesis of primary open-angle glaucoma and offers novel perspectives in the treatment of this serious disease. [ABSTRACT FROM AUTHOR]

There is an ongoing controversy regarding the role of WDR36 sequence variants in the pathogenesis of primary open-angle glaucoma (POAG). WDR36 is a nucleolar protein involved in the maturation of 18S rRNA. The function of WDR36 is essential as homozygous Wdr36 -deficient mouse embryos die before reaching the blastocyst stage. Here we provide a detailed analysis of the phenotype of heterozygous Wdr36 -deficient mice. Loss of one Wdr36 allele causes a substantial reduction in the expression of Wdr36 mRNA. In the eyes of Wdr36 +/− animals, the structure of the tissues involved in aqueous humor circulation and of the optic nerve head are not different from that of control littermates. In addition, one-year-old Wdr36 +/− animals do not differ from wild-type animals with regards to intraocular pressure and number of optic nerve axons. The susceptibility of retinal ganglion cells to excitotoxic damage induced by NMDA is similar in Wdr36 +/− and wild-type animals. Moreover, the amount of optic nerve axonal damage induced by high IOP is not different between Wdr36 +/− and wild-type mice. Transgenic overexpression of mutated Del605-607 Wdr36 in Wdr36 +/− animals does not cause changes in the number of optic nerve axons or susceptibility to excitotoxic damage. In addition, analysis of 18S rRNA maturation in Del605-607 Wdr36 +/− or Wdr36 +/− mice does not show obvious differences in rRNA processing or in the amounts of precursor forms when compared to wild-type animals. Our data obtained in Wdr36 +/− mice do not support the assumption of a causative role for WDR36 in the pathogenesis of POAG. [ABSTRACT FROM AUTHOR]

Purpose: The trabecular meshwork (TM) outflow pathways of the aqueous humor show an increase in extracellular matrix in patients with primary open-angle glaucoma (POAG). The increase in TM extracellular matrix appears to be caused by transforming growth factor-β signaling and its downstream mediator connective-tissue growth factor (CTGF). Here we studied whether treatment with the prostaglandin F2α analog fluprostenol modulates the CTGF-mediated increase of the TM extracellular matrix. Methods: Human TM cells from 3 different donors were treated with CTGF (50 ng/mL) and/or fluprostenol (10−6 M and 10−7 M) and were analyzed by real-time reverse transcription polymerase chain reaction and Western blotting. Cell supernatants of the treated cells were analyzed by zymography. Results: Treatment with CTGF induced the expression and synthesis of CTGF, fibronectin, collagen type IV and VI, while treatment with fluprostenol alone had no effects. The effects of CTGF were blocked by 1-h pretreatment with fluprostenol in a dose-dependent manner. Treatment with fluprostenol or combined fluprostenol/CTGF induced the activity of matrix metalloproteinase 2 (MMP2) in TM cells, whereas treatment with CTGF alone had no effects on MMP2 activity. Conclusions: Fluprostenol blocks the fibrotic effects of CTGF on human TM cells and increases the activity of MMP2. Both effects have the distinct potential to attenuate a CTGF-mediated increase in TM extracellular matrix in patients with POAG and any effects on TM outflow resistance that may result from that. [ABSTRACT FROM AUTHOR]

Abstract: 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-β2 causes an induction of ECM deposition in cultured human TM cells and that TGF-β2 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-β2-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 24h with CTGF at concentrations of 2.5–100ng/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-β2–induced gene expression was investigated by transfection of immortalized HTM cells with CTGF-specific small interfering (si)RNA before TGF-β2 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 β1. 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-β2–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-β2 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. [Copyright &y& Elsevier]

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]