Human conjunctival epithelial cell responses to platelet-activating factor (PAF): signal transduction and release of proinflammatory cytokines.

PURPOSE: The aims of the study were to characterize the signal transduction responses to platelet-activating factor (PAF) and to monitor the downstream effects of PAF on the production of proinflammatory cytokines in human conjunctival epithelial cells (HCECs). METHODS: The generation of inositol phosphates ([(3)H]IPs) from [(3)H]phosphoinositide (PI) hydrolysis and the mobilization of intracellular calcium ([Ca(2+)](i)) were evaluated using ion exchange chromatography and Fura-2 fluorescence techniques, respectively. The production of the cytokines (interleukin-6 [IL-6], interleukin-8 [IL-8], and granulocyte macrophage colony-stimulating factor [GM-CSF]) from PAF-stimulated HCECs was quantified using specific ELISA assays. Specific PAF antagonists were used to study the pharmacological aspects of PAF actions in HCECs. RESULTS: PAF (100 nM) maximally stimulated PI turnover in HCECs by 2.3+/-0.02 fold (n=21) above basal levels and with a potency (EC(50)) of 5.9+/-1.7 nM (n=4). PAF or its stabilized analog, methyl carbamyl (mc)PAF (EC(50)=0.8 nM), rapidly mobilized [Ca(2+)](i), which peaked within 30-60 s and remained elevated for 3 min. PAF (10 nM-1 microM) stimulated the release of the proinflammatory cytokines, IL-6, IL-8, and GM-CSF, 1.4-3.5 fold above basal levels. The effects of PAF (100 nM) on PI turnover and [Ca(2+)](i) were potently antagonized by the PAF antagonists, 1-o-hexadecyl-2-o-acetyl-sn-glycero-3-phospho (N,N,N-trimethyl) hexanolamine (IC(50)=0.69 microM; K(i)=38 nM), methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethyl-pyridine-3,5-dicsrboxylate (PCA-42481; IC(50)=0.89 microM; K(i)=50 nM), rac-3-(N-octadecylcarbomoyl)-2-methoxy) propyl-(2-thiazolioethyl) phosphate (CV-3988; IC(50)=13 microM; K(i)=771 nM), and (+/-)-cis-3,5-dimethyl-2-(3-pyridyl)thiazolidin-4-one HCl (SM-10661; IC(50)=14 microM; K(i)=789 nM [n=3 for each antagonist]). PAF-induced production of IL-6, IL-8, and GM-CSF from HCECs was also blocked by these PAF antagonists (IC(50)=4.6- 8.6 microM). CONCLUSIONS: HCECs respond to PAF by generating IPs, mobilizing [Ca(2+)](i), and then secreting cytokines into the extracellular medium. These results suggest that HCECs may be key target cells for the PAF released from conjunctival mast cells following ocular allergic reactions. Therefore, HCECs in culture represent suitable in vitro models for the investigation of the role of PAF in human ocular allergic and inflammatory diseases and for the discovery of therapeutically useful PAF antagonists.

Increased expression of the WNT antagonist sFRP-1 in glaucoma elevates intraocular pressure.

Elevated intraocular pressure (IOP) is the principal risk factor for glaucoma and results from excessive impedance of the fluid outflow from the eye. This abnormality likely originates from outflow pathway tissues such as the trabecular meshwork (TM), but the associated molecular etiology is poorly understood. We discovered what we believe to be a novel role for secreted frizzled-related protein-1 (sFRP-1), an antagonist of Wnt signaling, in regulating IOP. sFRP1 was overexpressed in human glaucomatous TM cells. Genes involved in the Wnt signaling pathway were expressed in cultured TM cells and human TM tissues. Addition of recombinant sFRP-1 to ex vivo perfusion-cultured human eyes decreased outflow facility, concomitant with reduced levels of beta-catenin, the Wnt signaling mediator, in the TM. Intravitreal injection of an adenoviral vector encoding sFRP1 in mice produced a titer-dependent increase in IOP. Five days after vector injection, IOP increased 2 fold, which was significantly reduced by topical ocular administration of an inhibitor of a downstream suppressor of Wnt signaling. Thus, these data indicate that increased expression of sFRP1 in the TM appears to be responsible for elevated IOP in glaucoma and restoring Wnt signaling in the TM may be a novel disease intervention strategy for treating glaucoma.

Increased expression of serum amyloid A in glaucoma and its effect on intraocular pressure.

PURPOSE: To search for and validate potential molecular pathogenic mechanisms in the trabecular meshwork (TM) responsible for the elevated intraocular pressure (IOP) associated with glaucoma. METHODS: Gene chip arrays were used to identify differential gene expression in glaucomatous TM tissues. Serum amyloid A (SAA) upregulation was subsequently confirmed with quantitative PCR (QPCR) and ELISA. The effect of SAA on gene expression of cultured human TM cells was tested with gene chip arrays and verified with ELISA, and its effect on IOP was evaluated in the human ocular perfusion organ culture. RESULTS: Microarray analysis showed that the expression of SAA2 was increased in TM tissues from donors with glaucoma. This finding was subsequently confirmed by QPCR. The SAA mRNA levels were increased in glaucoma TM tissues by more than 5-fold (P < 0.05) and in cultured TM cells derived from donors with glaucoma by 25-fold (P < 0.05) compared with controls. SAA protein levels in the TM of glaucoma patients were also significantly (P < 0.05) elevated by 2.9-fold. Treatment of cultured human TM cells with recombinant SAA affected gene expression, including a 22-fold up-regulation of interleukin-8 (P < 0.001). SAA increased IOP by approximately 40% (P < 0.05) in the human ocular perfusion organ culture without any observable changes in the morphology of the tissues involved in aqueous outflow. CONCLUSIONS: These findings indicate that SAA, which is an acute-phase apolipoprotein that plays important roles in infection, inflammation, and tissue repair, may contribute to the pathogenic changes to the TM in glaucoma.

Effects of TGF-beta2, BMP-4, and gremlin in the trabecular meshwork: implications for glaucoma.

PURPOSE: The primary causative factor of primary open-angle glaucoma (POAG) is elevated intraocular pressure (IOP) due to increased aqueous humor (AH) outflow resistance, which is associated with morphologic and biochemical changes in the trabecular meshwork (TM). Patients with glaucoma have elevated levels of transforming growth factor (TGF)-beta2 in their AH, and TGF-beta has been shown to increase TM extracellular matrix (ECM) production. The bone morphogenetic protein (BMP) signaling pathway modifies TGF-beta signaling in several different tissues, and a prior study demonstrated that TM cells and tissues express members of the BMP gene family. The purpose of this study was to determine whether BMPs can alter TGF-beta2 signaling in the TM and whether there are defects in BMP signaling in glaucoma. METHODS: ELISA, Western immunoblot analysis, and immunohistochemistry were used to evaluate the expression of BMP proteins in TM cells and tissues. ELISA was used to determine the effects of TGF-beta2 and BMPs on TM fibronectin (FN) secretion. Gene expression was determined by gene microarrays and quantitative (q)PCR. Perfusion-cultured human anterior segments were used to study the effects of altered BMP signaling on IOP. RESULTS: The human TM synthesized and secreted BMP-4 as well as expressed BMP receptor subtypes BMPRI and BMPRII. TM cells responded to exogenous BMP-4 by phosphorylating Smad signaling proteins. Cultured human TM cells treated with TGF-beta2 significantly increased FN levels, and BMP-4 blocked this FN induction. The expression of BMP family genes in normal and glaucomatous TM cells was profiled and significant elevation of mRNA and protein levels of the BMP antagonist gremlin were found in glaucomatous TM cells. In addition, Gremlin was present in human aqueous humor and in the perfusate medium of perfusion-cultured human eyes. Gremlin blocked the negative effect of BMP-4 on TGF-beta-induction of FN. Recombinant Gremlin added to the medium of ex vivo perfusion-cultured human eye anterior segments caused the glaucoma phenotype of elevated IOP. CONCLUSIONS: These results are consistent with the hypothesis that, in POAG, elevated expression of Gremlin by TM cells inhibits BMP-4 antagonism of TGF-beta2 and leads to increased ECM deposition and elevated IOP.

TGFbeta2-induced changes in human trabecular meshwork: implications for intraocular pressure.

PURPOSE: Transforming growth factor (TGF)-beta2 levels are elevated in glaucomatous human aqueous humor. TGFbeta is a cytokine that alters extracellular matrix (ECM) metabolism, and excess ECM has been proposed to increase aqueous outflow resistance in the trabecular meshwork (TM) of glaucomatous eyes. This study was undertaken to investigate effects of TGFbeta2 on secretion of fibronectin and the protease inhibitor plasminogen activator inhibitor (PAI)-1 from human TM cell cultures and perfused human ocular anterior segments. METHODS: Total RNA was isolated from pooled human TM cell monolayers and used for a gene microarray expression analysis. Supernatants from treated human TM cells were analyzed by ELISA for fibronectin or PAI-1 content. TGFbeta2 effects on intraocular pressure (IOP) were evaluated in a perfused organ culture model using human anterior segments, and eluates were analyzed for fibronectin and PAI-1 content. RESULTS: Overnight treatment of TM cells with TGFbeta2 upregulated multiple ECM-related genes, such as PAI-1. TGFbeta2 also increased secretion of both fibronectin and PAI-1 from TM cells. TGFbeta2 effects on TM cells were blocked by inhibitors of the TGFbeta type I receptor. In perfused human anterior segments, TGFbeta2 treatment elevated IOP and increased eluate fibronectin and PAI-1 content. CONCLUSIONS: TGFbeta2 effects on IOP may be transduced by TGFbeta type-I receptor-mediated changes in TM secretion of ECM-related factors such as fibronectin and PAI-1. Modulation of TGFbeta2-induced changes in the ECM may provide a novel and viable approach to the management of glaucoma.

Expression of matrix metalloproteinases and their inhibitors in human trabecular meshwork cells.

PURPOSE: Matrix metalloproteinases (MMPs) are involved in trabecular meshwork (TM) extracellular matrix metabolism and have been shown to increase aqueous outflow facility. The purpose of this study was to characterize effects of cytokines, a phorbol ester, and prostanoids on the expression of MMP-1, -2, -3, and -9 and tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in cultured human TM cells. METHODS: Five human TM cell strains were treated with selected compounds. Levels of proMMPs and TIMPs in cell media were quantified by ELISA. MMP-3 activity was assayed by casein zymography. RESULTS: All human TM cell strains produced detectable basal amounts of proMMPs and TIMPs. 12-O-tetradecanoyl-phorbol-13-acetate was effective in increasing the levels of proMMP-1, -3, and -9 and TIMP-1. Its effect on proMMP-1 was concentration-dependent with an EC(50) of 2 to 3 nM. Interleukin (IL)-1alpha did not affect levels of proMMP-1 and -2 or the TIMPs, but was most efficacious in increasing proMMP-3 production with an EC(50) of 0.5 ng/mL. The IL-1alpha-induced upregulation of proMMP-3 correlated with an increase in MMP-3 activity. Tumor necrosis factor-alpha activated proMMP-3 production in some but not all cell strains. Platelet-derived growth factor-BB was generally ineffective in modulating MMP and TIMP levels. Prostaglandins E(2) and F(2alpha) at 10 micro M did not affect levels of proMMP-1 or -3. CONCLUSIONS: The expression of the different MMPs and TIMPs in human TM cells was independently regulated. Production of MMP-3 was maximally activated by IL-1alpha. The IL-1alpha-stimulated expression of MMP-3 provides a probable mechanism for IL-1alpha-enhanced aqueous outflow.

Aqueous outflow-enhancing effect of tert-butylhydroquinone: involvement of AP-1 activation and MMP-3 expression.

PURPOSE: To test the effect of stimulators of activator protein (AP)-1, on expression of stromelysin (MMP-3) in human TM cells and on aqueous outflow in perfused human anterior segments. METHODS: Change in MMP-3 expression was determined by immunoassay of proMMP-3 levels in the media of cultured human TM cells. Anterior segments of human donor eyes with or without glaucoma were perfused with vehicle or the AP-1 stimulator tert-butylhydroquinone (tBHQ). The outflow rates or intraocular pressure (IOP), and proMMP-3 levels in the perfusate were monitored. RESULTS: AP-1 stimulators, such as beta-naphthoflavone, 3-methylcholanthrene, and tBHQ, significantly upregulated (2-4-fold) TM cell expression of MMP-3. The stimulatory effect of tBHQ was concentration dependent, with an EC(50) of approximately 3 micro M, and was blocked by concomitant treatment with 100 nM SR11302, which sequesters AP-1. When nonglaucomatous human eyes were perfused with tBHQ (10 micro M), both outflow rates and perfusate proMMP-3 level increased significantly within the first 24 hours. The outflow effect of tBHQ was suppressed when SR11302 (100 nM) was added in the perfusate. tBHQ also lowered the IOP by more than 40% in perfused glaucomatous eyes. CONCLUSIONS: An AP-1 activator, tBHQ, upregulated expression of MMP-3 in cultured human TM cells and perfused human eyes and enhanced outflow ex vivo. These effects were blocked by sequestering AP-1, suggesting that activation of AP-1 can lead to increased MMP-3 production in the TM, which in turn improves outflow facility. This unique mechanism may provide a novel therapy for glaucoma.