Comparison of cytokine/chemokine levels in aqueous humor of primary open-angle glaucoma patients with positive or negative outcome following trabeculectomy.

We aimed to identify differences in cytokine/chemokine levels in the aqueous humor (AH) of primary open-angle glaucoma (POAG) patients who suffered from scarring, compared with POAG patients with no scarring after trabeculectomy surgery. Identification of differently expressed cytokines and chemokines may help to understand scarring and fibrotic processes following trabeculectomy, and to make predictions for the outcome of fistulating surgery in the future. Furthermore, the identification of cell signaling pathways involved in fibrosis offers the opportunity for a more specific antifibrotic therapy with reduced side effects, and an improvement in long-term surgical outcome.Eight samples of AH were collected during trabeculectomy surgery and commercially available cytokine/chemokine arrays were used. Specific, differently expressed proteins (cytokines/chemokines) in AH samples from patients with positive and negative surgery outcomes were detected. These proteins were classified based on their known profibrotic, inflammatory, adhesive, and apoptotic properties. Transforming growth factor ß (TGF-ß) and vascular endothelial growth factor (VEGF) were among the most important profibrotic cytokines that we detected. Differences in the fold change of protein expression were highly significant between patients after successful and failed trabeculectomy surgery, and these were processed and visualized using ExprEssence software.This pilot study revealed differences in concentrations of cytokines/chemokines in AH between the two examined groups of patients. Our findings suggest that a positive outcome from trabeculectomy is strongly related to an inhibition of the fibrosis process.

PAX6 Expression and Retinal Cell Death in a Transgenic Mouse Model for Acute Angle-Closure Glaucoma.

PURPOSE: PAX6 is a highly conserved protein essential for the control of eye development both in invertebrates and vertebrates. PAX6 expression persists in the adult inner retina, but little is known about its functions after completion of retinal differentiation. Therefore, we investigated PAX6 expression in wild-type and calcitonin receptor-like receptor transgenic (CLR(SMαA)) mice with angle-closure glaucoma. METHODS: Intraocular pressure was measured by indentation tonometry in anesthetized mice. Eyes of mice of both genotypes were enucleated at various ages and retinas were processed for morphological analysis and PAX6 immunostaining. The content of PAX6 in retinal extracts was estimated by Western blot analysis. Retinal expression of glaucoma-related genes was analyzed by reverse transcription-polymerase chain reaction. RESULTS: Control mice showed normal retinal morphology between p22 and p428 with steady PAX6 expression in the ganglion cell layer (GCL) and the inner nuclear layer (INL). CLR(SMαA) mice examined between p22 and p82 exhibited increased intraocular pressure and a progressive decrease in cell number including PAX6-expressing cells in the GCL. The INL was not affected up to postnatal day 42. Later, a significant increase in PAX6-expressing cells concomitant with an overall loss of cells was observed in the INL of CLR(SMαA) as compared with control mice. Retinal up-regulation of glaucoma-related genes was furthermore observed. CONCLUSIONS: Distinctive changes of PAX6 expression in the inner retina of CLR(SMαA) mice suggest a role in regulatory mechanisms involved in glaucoma-related retinal cell death. The selective increase of PAX6 expression in the degenerating INL of CLR(SMαA) mice may represent an attempt to preserve retinal cytoarchitecture.

Constitutive expression of HCA(2) in human retina and primary human retinal pigment epithelial cells.

PURPOSE/AIM: HCA2, a receptor of ß-hydroxybutyrate and niacin, has recently been described in mouse retina and immortalized human retinal pigment epithelial (RPE) cell lines. As HCA2 might be a pharmacologic target, e.g. in diabetic retinopathy, we studied its expression in human retina and primary human RPE cells. MATERIALS AND METHODS: Paraffin sections of human retina and primary human RPE cells were obtained from human donor eyes. Expression of HCA2 in human retina was investigated by immunohistochemistry of paraffin sections and by RT-PCR. HCA2 expression in primary human RPE cells was examined by immunocytochemistry and by Western-blot analysis. RESULTS: Positive immunohistochemical staining for HCA2 was found in paraffin sections of human retina, and positive immunocytochemical staining for HCA2 in primary human RPE cells. RT-PCR analysis detected mRNA expression of HCA2 in human retina. The expression of HCA2 protein was found in primary human RPE cells. CONCLUSIONS: Based on these results, HCA2 appears to be constitutively expressed in human retina and in primary human RPE cells. Although its functional role is still unknown, HCA2 may be potentially involved in the pathogenesis of various retinopathies and may offer a new therapeutic target.

Topical application of PPADS inhibits complement activation and choroidal neovascularization in a model of age-related macular degeneration.

Age-related macular degeneration (AMD) is the most common cause of blindness among the elderly. AMD patients have elevated levels of membrane attack complex (MAC) in their choroidal blood vessels and retinal pigment epithelium (RPE). MAC forms pores in cell membranes. Low levels of MAC result in an elevation of cytokine release such as vascular endothelial growth factor (VEGF) that promotes the formation of choroidal neovascularization (CNV). High levels of MAC result in cell lysis and RPE degeneration is a hallmark of advanced AMD. The current standard of care for CNV associated with wet AMD is intravitreal injection of anti-VEGF molecules every 4 to 12 weeks. Such injections have significant side effects. Recently, it has been found that membrane pore-forming proteins such as α-haemolysin can mediate their toxic effects through auto- and paracrine signaling and that complement-induced lysis is amplified through ATP release followed by P2X receptor activation. We hypothesized that attenuation of P2X receptor activation may lead to a reduction in MAC deposition and consequent formation of CNV. Hence, in this study we investigated topical application of the purinergic P2X antagonist Pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) as a potential treatment for AMD. We found that 4.17 µM PPADS inhibited formation of HUVEC master junctions and master segments by 74.7%. In a human complement mediated cell lysis assay, 104 µM PPADS enabled almost complete protection of Hepa1c1c7 cells from 1% normal human serum mediated cell lysis. Daily topical application of 4.17 mM PPADS for 3 days attenuated the progression of laser induced CNV in mice by 41.8% and attenuated the deposition of MAC at the site of the laser injury by 19.7%. Our data have implications for the future treatment of AMD and potentially other ocular disorders involving CNV such as angioid streaks, choroidal rupture and high myopia.

Biological effects of cigarette smoke in cultured human retinal pigment epithelial cells.

The goal of the present study was to determine whether treatment with cigarette smoke extract (CSE) induces cell loss, cellular senescence, and extracellular matrix (ECM) synthesis in primary human retinal pigment epithelial (RPE) cells. Primary cultured human RPE cells were exposed to 2, 4, 8, and 12% of CSE concentration for 24 hours. Cell loss was detected by cell viability assay. Lipid peroxidation was assessed by loss of cis-parinaric acid (PNA) fluorescence. Senescence-associated ß-galactosidase (SA-ß-Gal) activity was detected by histochemical staining. Expression of apolipoprotein J (Apo J), connective tissue growth factor (CTGF), fibronectin, and laminin were examined by real-time PCR, western blot, or ELISA experiments. The results showed that exposure of cells to 12% of CSE concentration induced cell death, while treatment of cells with 2, 4, and 8% CSE increased lipid peroxidation. Exposure to 8% of CSE markedly increased the number of SA-ß-Gal positive cells to up to 82%, and the mRNA expression of Apo J, CTGF, and fibronectin by approximately 3-4 fold. Treatment with 8% of CSE also increased the protein expression of Apo J and CTGF and the secretion of fibronectin and laminin. Thus, treatment with CSE can induce cell loss, senescent changes, and ECM synthesis in primary human RPE cells. It may be speculated that cigarette smoke could be involved in cellular events in RPE cells as seen in age-related macular degeneration.

Preventive effects of omega-3 and omega-6 Fatty acids on peroxide mediated oxidative stress responses in primary human trabecular meshwork cells.

Pathologic processes in glaucoma include increased apoptosis, accumulation of extracellular material in the trabecular meshwork and optic nerve, condensations of the cytoskeleton and precocious cellular senescence. Oxidative stress was shown to generate these alterations in primary ocular cells. Fatty acids omega-3 and -6 are alleged to constitute a prophylaxis against these deleterious effects. Here, we tested actual preventive effects omega-3 and -6 against peroxide induced stress responses in primary human trabecular meshwork cells. Changes of mitochondrial activity, proliferation, heat shock proteins, extracellular matrix components, and inflammatory markers were evaluated. Alterations of the cytoskeleton were evaluated by phalloidin labeling. Here we report a repressive effect of omega-6 on metabolic activity and proliferation, which was not detected for omega-3. Both agents were able to prevent the anti-proliferative effect of H2O2, but only omega-3 prevented metabolic repression. Expression of heat shock protein 27 was unaltered by both fatty acids, whereas heat shock protein 90 was significantly induced by both. Omega-6 increased fibronectin and connective tissue growth factor synthesis, as well as the amount of secreted fibronectin. Omega-3, instead, induced plasminogen activator inhibitor 1 synthesis. H2O2 further increased fibronectin production in omega-6 supplemented cells, which was not the case in omega-3 treated cells. H2O2 stimulation of plasminogen activator inhibitor 1 and connective tissue growth factor was repressed by both fatty acids. Both fatty acids appeared to abolish H2O2 mediated stimulation of nuclear factor κB and IL-6, but not IL-1α and IL-8. H2O2 induced formation of cross-linked actin networks and stress fibers, which was reduced by preemptive application of omega-3. Omega-6, in contrast, had no protective effect on that, and even seemed to promote condensation. Based on the observed side effects of omega-6, omega-3 appears to be the more beneficial fatty acid in respect of prophylactic intake for prevention of a glaucomatous disease.

SPOC1 (PHF13) is required for spermatogonial stem cell differentiation and sustained spermatogenesis.

SPOC1 (PHF13) is a recently identified protein that has been shown to dynamically associate with somatic chromatin, to modulate chromatin compaction and to be important for proper cell division. Here, we report on the expression of SPOC1 in promyelocytic leukaemia zinc finger (PLZF)-positive undifferentiated spermatogonial stem cells (SSCs) of the mouse testis. To investigate further the biological function of SPOC1 in germ cells we generated Spoc1 mutant mice from a gene-trap embryonic stem cell clone. Postpubertal homozygous Spoc1(-/-) animals displayed a pronounced progressive loss of germ cells from an initially normal germ epithelium of the testis tubules leading to testis hypoplasia. This loss first affected non-SSC stages of germ cells and then, at a later time point, the undifferentiated spermatogonia. Remarkably, successive loss of all germ cells (at >20 weeks of age) was preceded by a transient increase in the number of undifferentiated A(aligned) (A(al)) spermatogonia in younger mice (at >10 weeks of age). The number of primary Spoc1(-/-) gonocytes, the proliferation of germ cells, and the initiation and progression of meiosis was normal, but we noted a significantly elevated level of apoptosis in the Spoc1(-/-) testis. Taken together, the data argue that SPOC1 is indispensable for stem cell differentiation in the testis and for sustained spermatogenesis.

Cytokine-dependent ELAM-1 induction and concomitant intraocular pressure regulation in porcine anterior eye perfusion culture.

PURPOSE: To demonstrate the capacity of interleukin (IL)-1 to simultaneously lower intraocular pressure (IOP) and induce trabecular ELAM-1 expression in one experiment and to test for IL-6 accordingly and evaluate the role of transforming growth factor (TGF)-ß2 as an IL-1 antagonist. METHODS: Forty-two porcine eyes were subjected to trabecular meshwork (TM) perfusion with IL-1α, -1ß, or -6 for 48 hours. Twelve of the IL-1α-treated eyes also received TGF-ß2 for the final 24-hour period. Polymerase chain reaction and Western blot analyses of ELAM-1 expression were then performed on harvested TM samples. mRNA regulation of IL-1α, IL-1ß, IL-6, TGF-ß2, and P-selectin (SELP) was determined. RESULTS: IL-1α and -1ß treatment augmented outflow facility approximately threefold while inducing ELAM-1. IL-6 perfusion neither changed IOP nor induced ELAM-1. IL-1α/TGF-ß2 double treatment significantly counteracted the IL-1-induced IOP decrease and markedly reduced the degree of ELAM-1 mRNA upregulation from 22.3- to 3.1-fold, and ELAM-1 protein from 1.9- to 1.2-fold. IL-1α mRNA was upregulated 5.3-, 3.3-, and 5.5-fold after perfusion with IL-1α, -1ß, and -1α/TGF-ß2, respectively. The respective values for IL-6 mRNA were 2.0-, 2.1- and 2.4-fold. Expression of IL-1ß and TGF-ß2 mRNA remained unchanged. IL-6 perfusion had no discernible regulatory effect. CONCLUSIONS: Simultaneous demonstration of IL-1's lowering of IOP and inducing trabecular ELAM-1 was achieved for the first time in one experiment, and its possible implications in the pathogenesis of glaucoma was further emphasized. The involvement of an autocrine feedback loop was confirmed in the porcine system. TGF-ß2 constitutes a potent IL-1 antagonist for IOP and ELAM-1 regulation.

Changes of osteopontin in the aqueous humor of the DBA2/J glaucoma model correlated with optic nerve and RGC degenerations.

PURPOSE: To identify age-dependent regulated aqueous humor (AH) factors in DBA2/J (D2J) mice and to correlate them with optic nerve degeneration and intraocular pressure (IOP) by population and individual analysis. METHODS: AH samples of D2J mice aged 2 (n = 3), 7 (n = 5), and 10 months (n = 14) were analyzed by mouse cytokine antibody array. Ten-month samples were classified into eyes with (D2J+) or without (D2J-) optic neuropathy. Ten-month-old C57/Bl6 (B6; n = 13) and DBA2/Rj (D2Rj; n = 15) mice served as controls. IOP was recorded from 2 to 10 months. Individual AH osteopontin (OPN) was determined in 31 D2J eyes (10 months) and was correlated with optic neuropathy and IOP. OPN mRNA was detected by in situ hybridization. OPN blood plasma content of D2J and B6 was monitored from 8 to 10 months. Effect of OPN on cell survival in the ganglion cell layer (GCL) or metabolism was tested in ex vivo-cultured D2Rj eyes and murine neuronal precursors. RESULTS: In array analysis, OPN was detected in 10-month-old D2J mice only. They significantly differed between D2J- and D2J+ (P = 0.006). By Western blot analysis, a sevenfold OPN increase in D2J+ was determined compared with B6. Individual analysis confirmed the positive correlation of OPN with optic neuropathy. IOP was not correlated with OPN. OPN blood plasma contents steadily increased with age in D2J. OPN(+) cells were detected within the ciliary body of D2J, and OPN(+) RGCs were ≈30% reduced. OPN treatment inhibited cell degeneration within the GCL in ex vivo-cultured D2Rj eyes and increased the metabolic activity of neuronal precursor cells. CONCLUSIONS: OPN is an age-dependent increased AH factor associated with degeneration of the optic nerve in D2J mice. By modulating the metabolism of neuronal cells, deregulated levels of OPN could be involved in degenerative processes affecting RGCs or optic nerve axons in the D2J model.

TGF-{beta}2 induces senescence-associated changes in human trabecular meshwork cells.

PURPOSE: Recent studies have revealed an accumulation of senescent cells in the outflow pathways in primary open-angle glaucoma (POAG). Transforming growth factor (TGF)-ß2 is thought to be involved in the pathologic changes of the trabecular meshwork (TM) of POAG eyes. The goal of this study was to determine whether TGF-ß2 triggers senescence-associated changes in human TM cells in vitro. METHODS: Cultured human TM cells were exposed to 1.0 ng/mL TGF-ß2 for 12, 24, and 48 hours. Senescence-associated ß-galactosidase (SA-ß-Gal) activity was investigated by histochemical staining. Lipid peroxidation was assessed after TGF-ß2 treatment. Levels of apolipoprotein J (Apo J), SM22, and osteonectin (SPARC) mRNA were determined by real-time PCR analysis. Furthermore, the effects of antioxidants on these TGF-ß2-mediated changes were tested. Induction of senescence-related signal transduction proteins (p16, p21, and pRb) was examined by real-time PCR and Western blot analysis. RESULTS: TGF-ß2 increased SA-ß-Gal activity, lipid peroxidation, and the mRNA expressions of Apo J, SM22, and SPARC. These TGF-ß2-induced changes were attenuated by antioxidants. TGF-ß2 increased p16 mRNA and protein expression, which was paralleled by a downregulation of pRb protein. There was no effect on p21 mRNA and protein expression after exposure to TGF-ß2. CONCLUSIONS: TGF-ß2 induces senescence-associated TM changes and activates the senescence-related p16-pRb signal transduction pathway in vitro. Thus, minimizing TGF-ß2 levels may help to prevent the ageing process in the TM as seen in POAG.

Expression of podoplanin and other lymphatic markers in the human anterior eye segment.

PURPOSE: To investigate the expression of lymphatic endothelial cell (LEC) markers in tissues of the anterior eye segment. METHODS: Sections of human anterior segments from eight eyes of eight donors (37-100 years) were stained for Vegf-R3, Prox-1, Lyve-1, Pdpn, Pcx, CCR7, Ccl19, Ccl21, and CD68. Pdpn localization was additionally analyzed by immunogold labeling on sections of five eyes. Expression of LEC markers and chemokine receptor/ligands was analyzed by RT-PCR in iris and trabecular meshwork (TM) tissue from three eyes and eight human TM (hTM) cell cultures. RESULTS: Vegf-R3 and Prox-1 stained no structures in the anterior segment. Lyve-1 stained single dendriform cells in the ciliary body, the TM, and the iris. Pdpn stained all trabecular cells, the cells of the trabeculum ciliare, and the anteriormost perimysium cells of the ciliary muscle. Schlemm's canal endothelium was not stained but reacted to a podocalyxin antibody. In the iris stroma, single dendriform cells were stained; at the anterior surface, almost all cells were Pdpn(+). Few stromal cells were Pdpn(+)/Lyve(+), but several anterior surface cells were Pdpn(+)/Ccl21(+). Solitary CCR7(+) cells were observed there, too. IF results were confirmed by PCR, but Prox-1 was detected in TM and iris. Cultured hTM cells displayed partial Pdpn/Ccl21 colocalization. CONCLUSIONS: Coexpression of Pdpn and Ccl21 at the anterior iris surface and in the chamber angle suggests the constitution of a chemokine gradient guiding APCs through the anterior chamber. The more pronounced expression of Pdpn in the TM could favor egression of APCs by way of the conventional outflow.