Activation of extracellular signal-regulated kinase in trabecular meshwork cells.

A number of different agents, such as growth factors, cytokines and phorbol esters have been shown to modulate trabecular meshwork cell function. These studies were designed to evaluate the role extracellular signal-regulated kinase (ERK) pathway plays in mediating the responses to platelet-derived growth factor-BB (PDGF-BB) and phorbol 12-myristate 13-acetate (PMA) in trabecular meshwork cells. The human trabecular meshwork cell line, HTM-3, and the bovine trabecular meshwork (BTM) cells were treated with either PDGF-BB or PMA and the activation of ERK 1/2 evaluated. The effects of the MAP kinase kinase (MEK) inhibitor U0126, and the PKC inhibitor chelerythrine on ERK 1/2 were also determined. In a separate group of experiments, cells were treated with PDGF-BB or PMA and the secretion of matrix metalloproteinase-2 (MMP-2) evaluated. The addition of PDGF-BB or PMA produced time- and dose-dependent activation of ERK 1/2. Pretreatment with U0126 or chelerythrine significantly reduced ERK 1/2 activation induced by PDGF-BB or PMA. The addition of PDGF-BB or PMA stimulated the secretion of MMP-2. This secretory response was inhibited by pretreatment with the MEK inhibitor U0126. In trabecular meshwork cells, PDGF-BB and PMA activate ERK 1/2 by a PKC-dependent mechanism. Activation of ERK 1/2 by these agents in trabecular meshwork cells leads to the secretion of MMP-2. These studies provide evidence that ERK pathway is an important mechanism for integrating various signals that regulate trabecular function.     

Bradykinin activation of extracellular signal-regulated kinases in human trabecular meshwork cells

Bradykinin stimulation of B₂ kinin receptors has been shown to promote matrix metallo-proteinase (MMP) secretion from trabecular meshwork cells and to increase conventional outflow facility. Because acute secretion of MMPs can be dependent on the activity of extracellular signal-regulated MAP kinases (ERK1/2), experiments were performed to determine bradykinin effects on ERK1/2 in cultured human trabecular meshwork cells and the relationship of these effects to MMP-9 release. Treatment of cells with bradykinin produced a rapid 4-to 6-fold increase in ERK1/2 phosphorylation. Stimulation of ERK1/2 activity peaked within 2 min and then declined to control levels by 60 min. The response maximum occurred with 100 nM bradykinin and the estimated EC₅₀ was 0.7 nM. Treatment of cells with the B₂ kinin receptor agonist, Tyr⁸- bradykinin, also stimulated ERK1/2 phosphorylation while the B₁ agonist, Lys- [Des-Arg⁹]- bradykinin had no significant effect. In addition, activation of ERK1/2 by bradykinin or Tyr⁸- bradykinin was blocked by the selective B₂ receptor antagonist, Hoe-140. Inhibition of MAP kinase kinase (MEK) with U0126 also blocked bradykinin-induced ERK1/2 phosphorylation. Suppression of protein kinase C activity with the nonselective inhibitor, GF109203X, or by down-regulation with phorbol ester, diminished, but did not eliminate, bradykinin activation of ERK1/2. A similar decrease of ERK1/2 stimulation was observed when Src kinase was inhibited by 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Finally, blockade of bradykinin-induced ERK1/2 activation substantially reduced the peptide’s action to stimulate MMP-9 release into the extracellular environment. The data demonstrate that bradykinin promotes ERK1/2 activation in human trabecular meshwork cells. The effect is mediated by B₂ kinin receptors, involves two different signaling pathways, and results in increased secretion of MMP-9.     

Modulation of conventional outflow facility by the adenosine A1 agonist N6-cyclohexyladenosine

PURPOSE: Studies have shown that the activation of adenosine A(1) receptors lower intraocular pressure primarily by increasing total outflow facility. The purpose of this study was to investigate the actions of the adenosine A(1) agonist N(6)-cyclohexyladenosine (CHA) on conventional outflow facility. METHODS: Conventional outflow facility was evaluated in isolated bovine anterior segments, perfused at a constant pressure of 10 mm Hg. After overnight perfusion to establish a stable baseline, the concentration- and time-dependent changes in outflow facility induced by CHA were determined. To confirm the involvement of adenosine A(1) receptors and matrix metalloproteinases (MMP) in any change in facility, the responses to CHA were evaluated in preparations treated with the adenosine A(1) receptor antagonist, 8-cyclopentyl-1,3-dimethylxanthine (CPT), or the nonselective MMP inhibitor GM-6001. RESULTS: The administration of CHA (10 microM) to perfused anterior segments produced a 28% increase in outflow facility over basal levels. This response was relatively slow to develop with no significant change in outflow facility measured until after 60 minutes of CHA infusion. The peak response to CHA infusion occurred between 3 and 4 hours after CHA administration. Analysis of the CHA concentration-response curves demonstrated that this increase in outflow facility was concentration-dependent, with an EC(50) of 0.28 microM. Pretreatment with the adenosine A(1) receptor antagonist CPT (10 microM) or the nonselective MMP inhibitor GM-6001 (10 microM) blocked the response to CHA (1 microM). When compared with control eyes, no significant change in baseline facility was measured in eyes perfused with CPT or GM-6001. CONCLUSIONS: These studies demonstrate that the adenosine agonist CHA significantly increases conventional outflow facility in the perfused bovine eye. Analysis of the CHA concentration-response curve and inhibition of the CHA-induced increase in outflow facility by the adenosine A(1) antagonist confirms that this response is mediated by the activation of adenosine A(1) receptors. The inhibition of the CHA-induced increase in outflow facility by the MMP inhibitor GM-6001 provides evidence that the secretion and activation of MMPs within the conventional outflow pathway play a central role in the ocular hypotensive action of adenosine A(1) agonists.     

白细胞介素-1α对猪小梁细胞基质金属蛋白酶／基质金属蛋白酶抑制剂表达的影响

背景房水外流通路的阻塞或小梁网细胞外基质（ECM）的异常堆积导致房水流畅系数降低是引起眼压升高的原因之一,基质金属蛋白酶（MMPs）和组织金属蛋白酶抑制剂（TIMPs）的平衡对ECM的代谢至关重要,白细胞介素-1α（IL-1α）可以通过调节MMPs的表达而调节房水的外流。目的研究猪重组IL-1α对体外培养的猪眼小梁细胞MMP-2、MMP-3及TIMP-1表达的影响。方法从猪眼取出带有小梁组织的巩膜,用组织块培养法培养小梁细胞并进行传代,第3代的猪小梁细胞应用纤维连接蛋白（FN）和层黏连蛋白（LM）进行鉴定。第3代小梁细胞血清饥饿培养24h后分为2组,对照组加入无血清培养基,IL组加入10m／L IL-1α,分别培养30min。采用细胞免疫化学法分析IL组MMP-2、MMP-3及TIMP-1蛋白在培养小梁细胞中的表达;采用逆转录聚合酶链反应（RT—PCR）法检测小梁细胞中MMP-2mRNA、MMP-3mRNA及TIMP-1 mRNA的表达,并与对照组的检测结果进行比较。结果传3代的细胞对FN和LM呈现阳性反应。与对照组比较,IL组小梁细胞中MMP-3和TIMP-1蛋白的表达量（A值）明显升高,差异均有统计学意义（t=-7．694、t=-5．199,P〈0．05）,但2组间小梁细胞中MMP-2表达的差异无统计学意义（t=-2．365,P〉0．05）。IL组小梁细胞中MMP-3mRNA和TIMP-1mRNA的表达量（A值）明显高于对照组,差异均有统计学意义（t=-3．025、t=-1．921,P〈0．05）,而2组间小梁细胞中MMP-2mRNA的表达差异无统计学意义（t=-1．173,P〉0．05）。结论外源性IL-1α能增加猪眼小梁细胞中MMP-3、TIMP-1的表达,引起MMP-3／TIMP-1平衡改变,促进小梁网ECM的分解,增加房水外流,而对MMP-2的表达无影响。     

Flufenamic acid enhances current through maxi-K channels in the trabecular meshwork of the eye

PURPOSE: Flufenamic acid relaxes trabecular meshwork, a smooth muscle-like tissue involved in the regulation of ocular outflow in the eye. In this study, we attempted to determine if ionic channels are involved in this response. METHODS: Cultured human (HTM) and bovine (BTM) trabecular meshwork cells were investigated using the patch-clamp technique. RESULTS: In trabecular meshwork, flufenamic acid (10(-5) M) reversibly stimulated outward current to 406 +/- 71% of initial outward current level in BTM (n = 10) and 294 +/- 75% of initial current level in HTM (n = 12) in all cells investigated; no significant differences emerged. The response was dosage-dependent. Replacement of potassium in all solutions eliminated the response to flufenamic acid (n = 4, BTM). Blocking K(ATP ) channels with glibenclamide (10(-5) M, n = 6) and small-conductance calcium-activated potassium channels with apamin (10(-6) M, n = 5) had no effect. A direct effect on calcium channels could also not be detected. Blockage of the large-conductance calcium-activated potassium channel (maxi-K) by iberiotoxin (10(-7) M) suppressed 87 +/- 9% (n = 6; HTM) and 91 +/- 10% (n = 6; BTM) of the response. Depleting the cells of calcium did not significantly alter the response to flufenamic acid. CONCLUSIONS: Flufenamic acid stimulates maxi-K channels in trabecular meshwork of both human and bovine origin. This should lead to hyperpolarization, closure of L-type channels and lowered cytosolic calcium levels, possibly explaining the relaxation observed in response to this substance.     

Cross-linked actin networks (CLANs) in bovine trabecular meshwork cells

A cytoskeletal feature of human trabecular meshwork (HTM) cells in vitro and ex vivo is the presence of cross-linked actin networks (CLANs) that are abundant in a proportion of TM cells exposed to dexamethasone (DEX) and also in cells from glaucoma patients. We wished to determine whether CLANs were present in the bovine trabecular meshwork (BTM), whether they were similarly induced by dexamethasone and whether the structures were comparable to CLANs in HTM cells. Cultures of HTM and BTM cells and ex vivo dissections of BTM tissue were stained with phalloidin (F-actin) and propidium iodide (nuclei) and imaged by confocal microscopy, thereafter being subjected to image analysis. Some CLAN-like structures were identified in ex vivo BTM tissue cultured with and without DEX. However we found that BTM cells in culture produced abundant CLANs when exposed to DEX; comparable to the best response from HTM cells. The CLANs were of similar dimensions and morphology to those found in human cells and they had a similar half life of 2 or 3 days following the removal of DEX. This work demonstrates that BTM cells provide a suitable model for future investigations of CLAN formation and function.BTM cultures are sufficiently hardy to thrive in low serum and serum-free conditions so we were able to show that aqueous humor stimulates CLAN formation in the target cells. Future research is directed at identifying the aqueous component(s) responsible for CLAN production.     

Influence of actin cytoskeletal integrity on matrix metalloproteinase-2 activation in cultured human trabecular meshwork cells

PURPOSE: The goal of this study was to investigate the possible link between actin cytoskeletal integrity and the activation of matrix metalloproteinases (MMPs) in trabecular meshwork (TM) cells. METHODS: Primary human TM (HTM) cells treated with different actin cytoskeleton-interfering agents, including cytochalasin D, latrunculin A, ethacrynic acid (ECA), a Rho kinase inhibitor (Y-27632), and H-7 (serine/threonine kinase inhibitor), were examined for changes in actin cytoskeletal organization by phalloidin staining, MMP-2 activation by gelatin zymography, expression of MT1-MMP by quantitative real-time PCR analysis, levels of tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2), and activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated protein kinase (ERK) by immunoblotting. RESULTS: Treatment of HTM cells with cytochalasin D and latrunculin A led to significant activation of MMP-2, p38 MAPK, and ERK1/2, which appeared to correlate with changes in cell morphology and actin depolymerization. Additionally, treatment with these cytoskeleton-disrupting agents elicited increased expression of MT1-MMP in HTM cells, concomitant with a decrease in the levels of secreted TIMP-1 and TIMP-2. In contrast, treatment with ECA, Y-27632, or H-7 triggered changes in cell shape and reduced actin stress fibers in HTM cells but did not exert significant effects on MMP-2 activation or MT1-MMP expression. CONCLUSIONS: These studies indicate that cytochalasin D- and latrunculin A-induced alteration of actin cytoskeletal integrity in HTM cells is associated with MMP-2 activation, most likely through the upregulation of its activator, MT1-MMP. These data provide a mechanistic connection between actin cytoskeletal organization and MMP-2 activation in TM cells and offer new insights into extracellular matrix remodeling in the aqueous outflow pathway.     

Selenium's effects on MMP-2 and TIMP-1 secretion by human trabecular meshwork cells

PURPOSE: Because of the observed increase in incidence of glaucoma among some individuals taking selenium as a dietary supplement, the present study was undertaken to investigate mechanisms of selenium-induced changes in homeostasis of human trabecular meshwork (HTM) cells. Specifically, the impact of selenium on matrix metalloproteinases (MMPs), their inhibitors (tissue inhibitors of metalloproteinases; TIMPs), and the second messengers that regulate MMP expression was investigated in an HTM cell culture model. METHODS: HTM cell cultures were treated with an organic selenium compound (methyl seleninic acid), and changes in secretion and activity of MMPs and TIMPs were analyzed by Western blot and zymography. Changes in extracellular-signal-related kinases 1 and 2 (ERK1/2) and phospho-ERK1/2 levels were monitored by Western blot analysis of whole-cell lysates prepared from selenium-treated cells. Photographs of cultures over time were used to document selenium-induced changes in cell morphology. RESULTS: Treatment of HTM cells with selenium for 24 hours at doses ranging from 1 to 10 micro M caused a dose-dependent decrease in the secretion of MMP-2 and TIMP-1. Treatment for 6 hours revealed a significant decrease in MMP-2 and TIMP-1 at the highest dose. MMP-1, -3, and -9 and TIMP-2 were either not detected or their secretion was not consistently influenced by selenium treatment. Selenium treatment caused a significant decrease in ERK1/2 phosphorylation, but no change in overall ERK protein levels. Selenium treatment resulted in dose-dependent, reversible changes in HTM cell-matrix associations. CONCLUSIONS: Selenium-induced changes in MMP-2/TIMP-1 secretion may alter the balance of extracellular matrix turnover in the conventional outflow pathway and cause an increase in intraocular pressure that eventually leads to glaucoma.     

Acute effects of PGF2alpha on MMP-2 secretion from human ciliary muscle cells: a PKC- and ERK-dependent process

PURPOSE: Studies were designed to evaluate the cellular mechanisms associated with prostaglandin (PG)F(2alpha)-induced matrix metalloproteinase (MMP)-2 secretion from human ciliary muscle (HCM) cells. METHODS: The secretion and activity of MMP-2 was determined by Western blot analysis and zymography, using conditioned medium and HCM cells. ERK1/2 activity was measured by in-gel kinase assay and Western blot analysis with anti-phospho-ERK1/2 antibodies. RESULTS: PGF(2alpha) increased the secretion of MMP-2 in a dose-dependent manner with an EC(50) of 2.7 x 10(-8) M. The addition of 1 muM PGF(2alpha) also increased MMP-2 secretion in a time-dependent manner with maximum secretion occurring at 4 hours after administration. At 4 hours, the maximum increase in MMP-2 secretion and activity were 112% +/- 32% and 88% +/- 18%, respectively. The secretory action of PGF(2alpha) was inhibited by pretreatment with a protein kinase C (PKC) inhibitor, chelerythrine chloride; the FP receptor antagonist, AL-8810; and the MEK inhibitor, PD-98059. The addition of PGF(2alpha) and latanoprost acid increased ERK1/2 activity by 117% +/- 12% and 75% +/- 9%, respectively. The PGF(2alpha)- and latanoprost-acid-induced ERK1/2 activation was blocked by the presence of PKC inhibitors and downregulation of PKC by prolonged incubation with a phorbol ester. CONCLUSIONS: These data provide evidence that FP receptor activation leads to an increase in the secretion and activation of MMP-2 through PKC- and ERK1/2-dependent pathways. FP-agonist-induced activation of ERK1/2 was blocked by PKC inhibitors, indicating that PKC activation is required for ERK1/2 activation and MMP-2 secretion from HCM cells. In the ciliary muscle, the functional responses to ERK1/2 activation include secretion of MMP-2, supporting the hypothesis that increases in uveoscleral outflow facility induced by PG administration involves the secretion and activation of MMP-2.     

莱菔硫烷对体外培养牛眼小梁细胞硫氧还蛋白表达的影响及其机制

背景 研究证实莱菔硫烷(SFN)可以激活多条通路,促进机体内抗氧化蛋白的表达.硫氧还蛋白(Trx)是维持细胞内氧化还原稳态的重要抗氧化蛋白之一. 目的 研究SFN对体外培养的牛眼小梁细胞内Trx表达的影响及其机制.方法体外培养牛眼小梁细胞并进行形态学观察,取第3代培养的牛眼小梁细胞,分别加入终浓度为0、10、20和30 μmol/L SFN培养30 min,应用实时荧光定量PCR技术检测细胞内Trx mRNA的表达.将细胞按照随机抽样的方法分为6个组,即空白对照组、LY294002组、U0126组、SFN组、LY294002+ SFN组和U0126+SFN组.采用Western blot法检测各组细胞中Nrf2蛋白和Trx蛋白的相对表达量.结果 体外成功培养牛眼小梁细胞.不同浓度SFN干预下,细胞Trx mRNA相对表达量总体比较差异有统计学意义(F=88.090,P＜0.01).LY294002+ SFN组、U0126+SFN组和SFN组细胞中Trx蛋白和Nrf2蛋白相对表达量显著高于空白对照组,差异均有统计学意义(均P＜0.01).LY294002+ SFN组和U0126+SFN组细胞中Trx蛋白和Nrf2蛋白相对表达量显著高于SFN组,差异均有统计学意义(均P＜0.01). 结论 SFN可通过磷脂酰肌醇-3激酶(PI3K)/蛋白激酶B(Akt)通路与丝裂原活化蛋白激酶(MAPK)/细胞外调节蛋白激酶1/2(ERK1/2)通路活化Nrf2,促进小梁细胞Trx mRNA及其蛋白的合成,保护细胞抵抗氧化应激损伤.     

Functional identification of phosphodiesterase activity in human trabecular meshwork cells.

The phosphodiesterases (PDE) activity in human trabecular meshwork cells (HTM-3) was investigated in this study in order to better understand the signal transduction pathways in the conventional outflow tract of the eye. Agonists (isoproterenol or nitroprusside) were used to stimulate adenylyl cyclase and guanylyl cyclase, respectively, in the absence and presence of nonselective IBMX or PDE5 specific inhibitors E4021 (1). The subcellular distribution of cAMP and cGMP PDEs was determined directly by PDE enzyme assays using HTM-3 cells. Levels of cyclic nucleotides were measured in the same cells by radioimmunoassay (RIA). Isoproterenol alone elevated cAMP levels, and this response was enhanced by IBMX. Nitroprusside alone caused no increase in basal cGMP levels but, in the presence of E4021, nitroprusside produced significant, dose-related elevation of cGMP levels. Subcellular distribution experiments indicated that the greatest activity for PDEs resided in the supernatant fraction. In conclusion, HTM-3 cells contain PDEs that degrade both cyclic nucleotides. The PDE activities reside predominantly in the supernatant, but the PDE activity for degrading cGMP is more pronounced. Moreover, results with E4021 suggest that PDE5 activity could play a critical role in modulating cGMP-related activity in the trabecular meshwork.     

Role of PKCepsilon in PGF2alpha-stimulated MMP-2 secretion from human ciliary muscle cells.

Studies were designed to examine the roles of individual protein kinase C (PKC) isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-induced matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Studies utilized primary cultures of human ciliary muscle cells. Individual PKC isoforms were detected by Western blotting, using PKC-isoform-specific antibodies. To evaluate MMP-2 secretion, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 4 h and the media analyzed for MMP-2 by Western blotting. To assess ERK1/2 activation, cells were serum-starved overnight, treated with PGF(2alpha) (1 micromol/L) for 5 min and cell lysates analyzed for ERK1/2 phosphorylation by Western blot analysis. To evaluate the roles of individual PKC isoforms, cells were pretreated with PKC inhibitors or siRNAs prior to the addition of PGF(2alpha). In cultured human ciliary muscle cells, the PKC isoforms exhibiting the highest level of expression were PKCalpha, epsilon, iota and lambda. The delta and eta isoforms exhibited moderate levels of expression and beta, gamma, and phi were not detected. The administration of PGF(2alpha) (1 micromol/L) primarily induced the translocation of PKCepsilon from cytosol to the membrane fraction, as well as increased MMP-2 secretion and ERK1/2 phosphorylation. The secretion of MMP-2 was inhibited by pretreatment with the broad-range PKC inhibitor, chelerythrine chloride; however, this response was not blocked by Go-6976, an inhibitor of conventional PKC isoforms. The PGF(2alpha)-induced secretion of MMP-2 was also blocked by pretreatment with the PKCepsilon-selective peptide translocation inhibitor, EAVSLKPT, or the transfection of siRNA-targeting PKCepsilon. The activation of ERK1/2 was inhibited by chelerythrine and the PKCepsilon translocation inhibitor. Human ciliary muscle cells express the alpha, epsilon, iota and lambda PKC isoforms. Stimulation of FP receptors in these cells activates PKCepsilon, resulting in ERK1/2 activation and an eventual increase in MMP-2 secretion. These data support the idea that the activation of FP receptors in vivo modulate uveoscleral outflow through the PKCepsilon-dependent secretion of MMPs.     

Human RPE-monocyte co-culture induces chemokine gene expression through activation of MAPK and NIK cascade

Cell-cell contact between human retinal pigment epithelium (hRPE) cells and monocytes occurs in many retinal diseases involving blood-retinal barrier breakdown. This study investigates chemokine secretion induced by co-culture of hRPE cells and monocytes and illustrates the roles of p38 kinase, ERK, JNK/SAPK and NF-kappaB-inducing kinase signaling pathways for hRPE IL-8 and MCP-1 secretion induced in hRPE by co-culture with monocytes. Co-culture of hRPE cells with monocytes increased steady-state IL-8 and MCP-1 mRNA and protein secretion. Stimulation of hRPE cells by monocytes resulted in prominent increases in p38, ERK1/2 and JNK/SAPK phosphorolation, IkappaBalpha degradation, and NF-kappaB nuclear translocation. The induced IL-8 and MCP-1 proteins were almost completely supporessed by U0126, a specific mitogen-activated protein kinase kinase (MEK) inhibitor, or by SB203580, a selective p38 inhibitor. Chemokine secretion was completely blocked by simultaneous administration of U0126 and SB203580. Induction of IL-8 and MCP-1 was abrogated by Ro318220, an inhibitor of PKC, as well as by genistein or herbimycin A, inhibitors of PTK. In addition, anti-inflammatory drugs dexamethasone (DEX) and cyclosporin A (CSA) both blocked activation of JNKS/SAPK and the cell-cell contact induced production of hRPE IL-8 and MCP-1, while activation of p38 and ERK was only inhibited by DEX, but not by CSA. These results suggest that activation of DEX-sensitive, CSA-resistant MEK/ERK and p38 pathways, and activation of NF-kappaB, PKC, and PTK are essential for IL-8 and MCP-1 expression by hRPE cells.     

Pilocarpine-induced lysosomal enzyme secretion,from bovine trabecular meshwork cells

It has long been held that pilocarpine-induced ciliary muscle contraction causes a mechanical change in the configuration of the trabecular meshwork, thereby increasing its fluid conductance. Though the results of many studies are consistent with this theory, other findings suggest a direct action of pilocarpine on the meshwork. We used cultured bovine trabecular meshwork (BTM) cells to study the cell biological effects of pilocarpine, chemically a weak base, and demonstrated increased vacuolization of the BTM cells after incubation with pilocarpine, reflecting a trapping of the protonated base inside the lysosomes. We also showed that pilocarpine enhanced the release of lysosomal hydrolases into the medium. We hypothesize that this is clinically relevant and that the pilocarpine-induced release of hydrolases modifies the extracellular matrix of the trabecular meshwork, thereby increasing its fluid conductance.     

Aqueous humor stimulates the migration of human trabecular meshwork cells in vitro

PURPOSE: Depletion of trabecular meshwork cell numbers is a feature of the outflow system in aging and in primary open-angle glaucoma. It is possible that migration stimulated by factors present in aqueous humor may contribute to the cell loss. This investigation assessed the chemoattractant potential of glaucomatous and nonglaucomatous human aqueous humor and fibronectin, one of its constituents, on a range of cultured trabecular meshwork cell lines. METHODS: Migration was assessed in 48-well modified Boyden chambers. The potential migratory stimulants were soluble fibronectin and glaucomatous and nonglaucomatous aqueous humor. The glaucomatous aqueous samples were collected from patients undergoing trabeculotomy for primary open-angle glaucoma and the normal aqueous from normal bovine eyes and patients undergoing cataract surgery. The target cell types were normal human and bovine meshwork cells grown from explants and two human transformed meshwork cell lines from a normal (HTM-5) and a glaucomatous (HTM-3) source. RESULTS: Soluble fibronectin stimulated all the target cells to migrate with an optimal concentration ranging from 1 to 30 microg/ml, and Zigmond Hirsch checkerboard analysis indicated that both chemotaxis and chemokinesis took place. All the aqueous humor samples stimulated migration of the meshwork cell lines at an optimal concentration of 200 microl/ml. Glaucomatous aqueous humor stimulated a greater migratory response than nonglaucomatous aqueous for two of the four target cell types (P < or = 0.03). Neutralization of the fibronectin content of nonglaucomatous and glaucomatous aqueous by addition of excess anti-fibronectin antibody indicated that fibronectin could account for 35% to 80% of the migratory activity of the aqueous. CONCLUSIONS: Aqueous humor contains potentially powerful chemoattractants for trabecular meshwork cells. The activity of one of these constituents, fibronectin, has been accounted for by this study. Glaucomatous aqueous appears to be as good and in some cases a better migratory stimulant than nonglaucomatous aqueous in vitro. The migratory evidence points to a trend that may help to explain cell loss in the aging meshwork and possibly some of the extra loss in primary open-angle glaucoma.     

内源性大麻素对体外培养的牛眼小梁细胞PGE2表达及细胞骨架的影响

目的:研究内源性大麻素N-花生四氢酸氨基乙醇(anan-damide,AEA)对体外培养的牛眼小梁细胞细胞骨架及PGE2表达的影响。方法:对牛眼小梁细胞进行原代及传代培养,并行鉴定。对传3代的牛眼小梁细胞分别施加AEA浓度分别为0,0.1,1.0,10μmol/L的无血清培养液,作用24h后提取细胞上清液,并将细胞置于倒置显微镜下摄像,计算机图像分析系统计算细胞面积。用ELISA法检测PGE2浓度的变化。结果:AEA可以产生明显的细胞舒张效应,呈浓度依赖性。而且在一定范围内可以成剂量依赖性的促进PGE2的表达。与对照组均有显著性差异(P<0.05)。结论:AEA可引起小梁细胞的舒张和促进PGE2的释放。     

Kinin modulation of conventional outflow facility in the bovine eye.

The aim of this study was to investigate the effects of bradykinin on conventional outflow facility in relation to kinin effects on matrix metalloproteinase (MMP) secretion. Conventional outflow facility was measured in isolated bovine segments perfused at a constant pressure of 10 mmHg. Experiments were also performed in primary cultures of bovine trabecular meshwork cells to assess bradykinin effects on the secretion of MMP-9 assessed by western blot. Administration of bradykinin (10(-7) M) to perfused anterior segments produced a 50% increase in outflow facility above basal levels. The effect was slow to develop, requiring 100 min for a significant increase in facility and 4 h for the peak response to be observed. Pretreatment of anterior segments with the B(2) kinin receptor antagonist, HOE-140 (10(-6) M), or with the nonselective MMP inhibitor, GM6001 (10(-5) M) blocked the response to bradykinin (10(-7) M). Treatment of cultured trabecular meshwork cells with bradykinin (10(-7) M) for 120 min stimulated secretion of MMP-9 into the extracellular media, and this response was inhibited by HOE-140 (10(-6) M). These results demonstrate that bradykinin activates B(2) kinin receptors to increase conventional outflow in the perfused bovine eye and provide evidence that secretion and activation of MMPs within the conventional pathway may mediate the effect.     

Bovine Trabecular Cells Produce TIMP-1 and MMP-2 in Response to Mechanical Stretching

Bovine trabecular cells in growth phase were exposed to cyclic mechanical stretching of the bottom of a culture dish at a cycle of 30 seconds for 72 hours. The stretched cells produced significantly larger amounts of metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-1 (TIMP-1) after 72 hours, compared with cells in nonstretched control. In contrast, TIMP-2 and MMP-9 levels were not influenced by mechanical stretching. Trabecular cells would modify extracellular matrix in response to such mechanical stimuli as bending of trabecular meshwork or aqueous flow by the production of TIMP-1 and MMP-2.