Bovine Trabecular Meshwork Cells Research Articles

α-Synuclein modulates fibronectin expression in the trabecular meshwork independent of TGFβ2

α-Synuclein (α-Syn) is implicated in Parkinson's disease (PD), a neuromotor disorder with prominent visual symptoms. The underlying cause of motor dysfunction has been studied extensively, and is attributed to the death of dopaminergic neurons mediated in part by intracellular aggregation of α-Syn. The cause of visual symptoms, however, is less clear. Neuroretinal degeneration due to the presence of aggregated α-Syn has been reported, but the evidence is controversial. Other symptoms including those arising from primary open angle glaucoma (POAG) are believed to be the side-effects of medications prescribed for PD. Here, we explored the alternative hypothesis that dysfunction of α-Syn in the anterior eye alters the interaction between the actin cytoskeleton of trabecular meshwork (TM) cells with the extracellular matrix (ECM), impairing their ability to respond to physiological changes in intraocular pressure (IOP). A similar dysfunction in neurons is responsible for impaired neuritogenesis, a characteristic feature of PD. Using cadaveric human and bovine TM tissue and primary human TM cells as models, we report two main observations: 1) α-Syn is expressed in human and bovine TM cells, and significant amounts of monomeric and oligomeric α-Syn are present in the AH, and 2) primary human TM cells and human and bovine TM tissue endocytose extracellular recombinant monomeric and oligomeric α-Syn via the prion protein (PrPC), and upregulate fibronectin (FN) and α-smooth muscle actin (α-SMA), fibrogenic proteins implicated in POAG. Transforming growth factor β2 (TGFβ2), a fibrogenic cytokine implicated in ∼50% cases of POAG, is also increased, and so is RhoA-associated coiled-coil-containing protein kinase 1 (ROCK-1). However, silencing of α-Syn in primary human TM cells reduces FN, α-SMA, and ROCK-1 in the absence or presence of over-expressed active TGFβ2, suggesting modulation of FN and ROCK-1 independent of, or upstream of TGFβ2. These observations suggest that extracellular α-Syn modulates ECM proteins in the TM independently or via PrPC by activating the RhoA-ROCK pathway. These observations reveal a novel function of α-Syn in the anterior eye, and offer new therapeutic options.

Effect of IL-6 on fibronectin in bovine trabecular meshwork cells cultured in vitro

Effect of IL-6 on fibronectin in bovine trabecular meshwork cells cultured <i>in vitro</i>

Expression changes of transforming growth factor-β1 and fibronectin in bovine trabecular meshwork cells cultured by iris pigment particles

Background Pigmentary glaucoma and pseudoexfoliation glaucoma are characterized by pigment dispersion in trabecular meshwork, and the dipersitional pigment probably contributes to the resistance of aqueous outflow pathway, irreversible damage of the trabecular meshwork and the remodeling abnormality of extracellular matrix.Researches determined that contents of transforming growth factor-β (TGF-β) in the aqueous humor are increased in glaucomatous eyes.However, the effects of TGF-β and fibronectin (FN) in the trabecular meshwork cells (TMCs) acted by iris pigmentary particles still are not elucidated. Objective This study was to investigate the effects of iris pigment particles on TGF-β1 and FN expression in bovine TMCs (BTMCs) cultured in vitro. Methods BTMCs were cultured in vitro by explant culture method and identified by morphological evaluation.The third generation of BTMCs were divided into normal control group and pigment group, and 100 μl PBS and 100 μl iris pigment suspension (final concentration of 1×107 particles/ml) were added into the medium for 24 hours, respectively.The expressions of TGF-β1 mRNA, FN mRNA and their proteins in the BTMCs were assayed by real-time fluorescence quantitative PCR and ELISA, respectively. Results Cultured cells grew well and showed the fusiform, polygon and dendritic like in shape, with pigmented and round nuclei.The relative expression levels of TGF-β1 mRNA and FN mRNA in the cells were 2.98±0.27 and 0.36±0.10 in the iris pigment group, which were significantly higher than 1.00±0.00 and 1.00±0.00 in the normal control group (t=12.68, 10.60, both at P=0.00). The concentrations of TGF-β1 protein and FN protein in the cell suspension were (156.60±9.74)ng/L and (59.29±15.79)mg/ml in the iris pigment group, showing significant differences in comparison with (65.46±14.24)ng/L and (102.10±12.14)mg/ml in the normal control group (t=9.15, P=0.00; t=3.72, P=0.02). Conclusions The expression of TGF-β1 is up-regulated and that of FN is down-regulated in BTMCs cultured by iris pigment, inferring that TGF-β1 and FN participate in the pathogenesis and development of pigmentary and pseudoexfoliation glaucoma. Key words: Glaucoma; Gene expression regulation; Iris/metabolism; Melanins/metabolism; Trabecular meshwork/metabolism; Transforming growth factor beta1/metabolism; Fibronectin; Cells, cultured

A Comparison of Gene Expression Profiles between Glucocorticoid Responder and Non-Responder Bovine Trabecular Meshwork Cells Using RNA Sequencing.

The most common ocular side effect of glucocorticoid (GC) therapy is GC-induced ocular hypertension (OHT) and GC-induced glaucoma (GIG). GC-induced OHT occurs in about 40% of the general population, while the other 60% are resistant. This study aims to determine the genes and pathways involved in differential GC responsiveness in the trabecular meshwork (TM). Using paired bovine eyes, one eye was perfusion-cultured with 100nM dexamethasone (DEX), while the fellow eye was used to establish a bovine TM (BTM) cell strain. Based on maximum IOP change in the perfused eye, the BTM cell strain was identified as a DEX-responder or non-responder strain. Three responder and three non-responder BTM cell strains were cultured, treated with 0.1% ethanol or 100nM DEX for 7 days. RNA and proteins were extracted for RNA sequencing (RNAseq), qPCR, and Western immunoblotting (WB), respectively. Data were analyzed using the human and bovine genome databases as well as Tophat2 software. Genes were grouped and compared using Student’s t-test. We found that DEX induced fibronectin expression in responder BTM cells but not in non-responder cells using WB. RNAseq showed between 93 and 606 differentially expressed genes in different expression groups between responder and non-responder BTM cells. The data generated by RNAseq were validated using qPCR. Pathway analyses showed 35 pathways associated with differentially expressed genes. These genes and pathways may play important roles in GC-induced OHT and will help us to better understand differential ocular responsiveness to GCs.

Protection of sulforaphane against hydrogen peroxide-induced bovine trabecular meshwork cell apoptosis

Background Evidences indicated that oxidative stress damage is an essential pathological process in primary open angle glaucoma.Sulforaphane (SFN) can play an antioxidative stress role to many tissues and cells by activating Nrf2/ARE single pathway.However, whether SFN has a protective role to oxidative stress induced damage of trabecular meshwork cells is still unclear. Objective This study was to investigate the antioxidant effect of SFN against H2O2-induced oxidative damage in bovine trabecular meshwork cells. Methods Trabecular cells were isolated from fresh black bovine eyeballs and primarily cultured and passaged.The third generation of cells were incubated to 96-well dish at a density of 1×103/well for 24 hours and divided into 4 groups.The cells were incubated using 100 μl serum-free medium in the blank control group.Oxidative damage models were established by adding 100 μmol/L H2O2 (100 μl) in medium in the H2O2 group.The cells were cultured with the medium containing 10 μmol/L SFN (100 μl) in the SFN group, and 100 μl H2O2at the final concentration of 100 μmol/L was added in the SFN-treated cell medium in the SFN+ H2O2 group.The cell vitality in various groups was assayed by using cell counting kit-8 (CCK-8). The apoptosis rate of the cells was detected by Annexin V-FITC/PI double-staining with flow cytometry. Results Cultured cells showed a spindle shape with uniform size, abundant cytoplasm, numberous pigmented particles and big nucleolus.The relative cell vitality reduced to (67.00±1.27)% and (80.00±6.25)% in the H2O2 group and SFN+ H2O2 group in comparison with 100% in the blank control group, and the cell vitality in the SFN+ H2O2 group was lower than that in the SFN group but higher than that in the H2O2 group (both at P<0.01). The mean apoptosis rate was (11.33±0.77)%, (32.31±1.03)%, (10.44±0.68)% and (17.68±0.21)% in the blank control group, H2O2 group, SFN group and SFN+ H2O2 group, respectively, showing a significant difference among the groups (F=539.96, P<0.01), and the apoptosis rate in the SFN+ H2O2 group was significantly lower than that in the H2O2 group but higher than that in the blank control group and SFN group (all at P<0.01). Conclusions SFN can improve the antioxidative stress ability of trabecular meshwork cells and alleviate the damage induced by oxidative stress. Key words: Isothiocyanates; Thiocyanates/pharmacology; Cells, cultured; Hydrogen peroxide/toxicity; Oxidative stress/drug effects; Trabecular meshwork cells/drug effects; Apoptosis; Animals, cattle; Sulforaphane

Characterization of a spontaneously immortalized bovine trabecular meshwork cell line

Trabecular meshwork (TM) cells have widely been used as an in vitro model for glaucoma research. However, primary TM cells suffer the disadvantages of limited cell numbers and slow rates of proliferation. We discovered a spontaneously transformed bovine TM (BTM) cell line, BTM-28T. This cell line proliferated rapidly in low-glucose culture medium but also demonstrated contact inhibition in high-glucose culture medium. BTM-28T cells expressed key TM cell markers including α-smooth muscle actin (α-SMA), laminin and collagen IV (col IV). Also, 100 nM dexamethasone (DEX) enhanced the formation of cross-linked actin networks (CLANs) in confluent BTM-28T cell cultures. Transforming growth factor beta 2 (TGFβ2) induced the expression of fibronectin (FN), plasminogen activator inhibitor-1 (PAI-1), and connective tissue growth factor (CTGF) in our cell cultures. This cell line will be helpful to better understand the aqueous humor outflow pathway as related to the pathophysiology of glaucoma.

Inducers of Cross-Linked Actin Networks in Trabecular Meshwork Cells

It is well established that the unusual actin arrangements known as cross-linked actin networks (CLANs) can be induced by dexamethasone (DEX) in trabecular meshwork (TM) cells. Recent work reporting their presence in elderly glaucomatous and nonglaucomatous tissue, however, has highlighted the presence of other inducers. In this study, the authors sought to identify CLAN induction agents that may be present within and around the outflow system. Studies were conducted on confluent bovine TM (BTM) cells in culture, and actin was stained with Alexa-Fluor 488 phalloidin to identify CLANs in the target cells. The CLAN-inducing potential of aqueous humor was expanded and included investigation of transforming growth factor-beta 2 (TGF-β2). The effect of decorin and fetal calf serum (FCS) on BTM cell cytoskeleton was also investigated, and all were compared with DEX with an exposure period of up to 7 days. CLAN numbers were increased after 7 days of exposure to TGF-β2 (45%), aqueous humor (37%), and decorin (69%). Even FCS had some modest CLAN-inducing ability (reaching 12%) in BTM cells. Neutralization of TGF-β2 reduced CLAN incidence in aqueous humor conditions to baseline (12%) levels. Blocking TGF-β2 receptors reduced CLAN formation in TM cells by 25% to 30%, whereas the inhibition of Smad3 negated CLAN incidence. In this study the authors identified TGF-β2 as a CLAN-inducing component present in aqueous humor. Decorin was also implicated as another CLAN-inducing agent and it was confirmed that FCS has CLAN-inducing properties.

Effect of Elevated Intracellular cAMP Levels on Actomyosin Contraction in Bovine Trabecular Meshwork Cells

Elevated cAMP in the trabecular meshwork (TM) cells increases the aqueous humor outflow facility. The authors investigated the mechanisms by which elevated cAMP opposes the RhoA-Rho kinase pathway, leading to the relaxation of the actomyosin system in bovine TM cells. Forskolin (Fsk) and rolipram were used to elevate cAMP levels. Changes in the phosphorylation of RhoA at Ser188 (a putative inhibitory site), the regulatory light chain of myosin (pMLC), and the regulatory subunit of myosin phosphatase (MYPT1) were determined by Western blot analysis. The actomyosin contraction was measured by collagen gel contraction (CGC) assay. The impact of cAMP on cell-matrix adhesion was followed by immunostaining of focal adhesion proteins and by electric cell-substrate impedance sensing. Elevated cAMP led to an increase in the phosphorylation of RhoA at Ser188, to the inhibition of endothelin-1 (ET-1)-induced activation of RhoA, and to the formation of stress fibers. The loss of pMLC along the stress fibers was comparable to that induced by Y-27632 (Rho kinase inhibitor). A concomitant reduction in both MYPT1 phosphorylation and pMLC was observed. Elevated cAMP also reduced (ET-1)-induced CGC and the cell-substrate resistance by >50%. In TM cells, elevated cAMP leads to the phosphorylation of RhoA at Ser188. Consequent inhibition of RhoA activity reduces the phosphorylation of MYPT1 at Thr853, leading to a reduction in MLC phosphorylation and actomyosin contraction. These actions, similar to those of the Rho kinase inhibitors, possibly underlie the reported increase in outflow facility in response to Fsk perfusion ex vivo.

Rapid and delayed death of cultured trabecular meshwork cells after selective laser trabeculoplasty

Selective laser trabeculoplasty (SLT) is becoming increasingly employed to reduce elevated intraocular pressure in glaucoma patients. SLT is known to target the ocular trabecular meshwork (TM), but the exact response mechanisms to this treatment have not been clearly delineated. The aim of the present study, therefore, was to investigate the modes of death of cultured bovine TM cells subjected to SLT in vitro. Bovine TM cell cultures were established, pigmented with exogenous melanin and irradiated with a Q-switched, frequency doubled, Nd:YAG laser, at different energy settings (0.05-1.0 mJ). Influences on cells were determined for up to 10 days post-treatment by trypan blue exclusion, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and by morphological assessment. Furthermore, homogeneous mixtures of pigmented and non-pigmented TM cells were irradiated to ascertain selectivity of laser effects. At higher energy levels (1.0, 0.75 mJ), immediate loss of cells was detected at the irradiated site. Trypan blue exclusion analysis showed that necrotic cell death subsequently occurred up to 8 hours following irradiation, peaking at 60 minutes. This was followed by delayed cell death peripheral to the irradiated area which was characteristic of apoptosis and which peaked at 2-3 days post-treatment. When mixed cultures were tested, laser treatment selectively killed pigmented cells at an energy level equivalent to the lower cell killing threshold in the initial studies (0.2 mJ) but at the higher laser energy of 0.35 mJ, all cells were non-selectively killed. SLT treatment killed pigmented TM cells in culture by a variety of processes (instant vaporization, rapid necrosis, delayed apoptosis), depending on the magnitude of the energy used and the distance from the center of the irradiated zone. These data may assist in the elucidation of the mechanism of action of the SLT procedure on TM cells in situ.

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.

Effects of Platelet-Derived Growth Factor on Aqueous Humor Dynamics

It is well known that the small GTPase RhoA modulates actin cytoskeleton and cellular contractility in the trabecular meshwork (TM). Several substances known to contract the TM reduce outflow facility, whereas cellular relaxation is commonly associated with the opposite effect. Inhibitors of the RhoA pathway are under development as antiglaucoma drugs. Here the authors investigate the role of platelet-derived growth factor (PDGF), a known activator of the Rac1 pathway, in cell cytoskeleton, outflow facility, and intraocular pressure (IOP). Effects of PDGF on actin cytoskeleton, Rac1, and AKT activation were tested in preconfluent and confluent bovine TM cells in culture. Rac1 and AKT/P-AKT activation were assessed by Western blot analysis. Trabecular outflow facility was measured in bovine perfused anterior segments. Changes in IOP were measured for up to 6 hours after topical application in the cornea of rabbit eyes by means of a contact tonometer. In TM cells, PDGF (10 ng/mL) activated Rac1 through AKT and induced actin cytoskeleton rearrangement with lamellipodia formation. In this sense, lamellipodia formation in TM cells was prevented by NSC23766, a Rac1 inhibitor, and LY294002, a PI3K inhibitor. In perfused anterior segments, PDGF (100 ng/mL) increased trabecular outflow facility by 26%. In vivo, when topically applied to rabbit corneas, PDGF induced a 20% decrease in IOP (100 ng/mL). This reduction was concentration dependent and presented an EC(50) value of 2.7 nM. PDGF, by activating the Rac1 pathway, induces cytoskeletal changes in TM cells that enhance outflow facility. Decreased IOP after PDGF application is likely caused by the facilitation of aqueous humor outflow. Rac1 pathway activation appears to be a positive modulator of outflow facility and an interesting target for decreasing IOP after ocular hypertension.

Effects of endothelin-1 on calcium-independent contraction of bovine trabecular meshwork

Endothelin-1 (ET-1) is known to induce contraction of trabecular meshwork (TM) and is probably involved in the pathogenesis of glaucoma. Calcium (Ca(2+))-independent contraction has been shown in TM, and its inhibition may represent an interesting way of influencing outflow facility, and thus intraocular pressure (IOP). This study investigates the role of ET-1 and its receptors ET-A and ET-B (ET-AR and ET-BR) in TM Ca(2+)-independent contractility. Isometric tension measurements of bovine TM (BTM) strips were performed using a force-length transducer system. Intra- and extracellular Ca(2+) buffering was achieved by means of EGTA and BAPTA-AM. Under Ca(2+)-free conditions, ET-1-induced contractility of TM was assessed also in the presence of the specific inhibitors for ET-AR and ET-BR, BQ123 and BQ788 respectively. In order to clarify the intracellular mediators of Ca(2+)-independent contractility induced by ET-1, TM contraction was further measured in the presence of Y-27632, a selective inhibitor of Rho-associated kinases (ROCKs). The expression of ROCK1 and of its activating protein RhoA in BTM cells was investigated using western blot analysis. ET-1 induced a significant contraction of native BTM after intra- and extracellular Ca(2+)-depletion (45% +/- 8% of the maximally inducible contraction). Both endothelin receptor inhibitors BQ123 and BQ788 significantly reduced TM Ca(2+)-independent contraction in response to ET-1 (8.4 +/- 3.3% and 20.3 +/- 4.8% respectively). In the presence of the ROCK inhibitor Y-27632, ET-1-induced contraction of TM under Ca(2+)-free conditions was almost completely abolished (4.3% +/- 1.7%, p < 0.001). A clear signal for RhoA at 24 kDa and ROCK1 at 160 kDa could be detected in lysates of native tissue and cultured BTM cells with western blot. This study shows evidence that a significant portion of ET-1-induced contraction of TM is Ca(2+)-independent. In this contraction pathway, both ET-AR and ET-BR are involved with RhoA and its kinases as intracellular mediators. Ca(2+)-independent contraction of TM in response to ET-1 may represent a specific target to modulate IOP.

Activation of Store-Operated Ca2+Channels in Trabecular Meshwork Cells

In nonexcitable cells, G(q)-coupled membrane receptor activation induces a biphasic increase in intracellular calcium ([Ca(2+)](i)) expressed as an initial IP(3)-dependent release from intracellular stores followed by a sustained Ca(2+) influx from the extracellular space that involves store-operated Ca(2+) channels (SOCs). In trabecular meshwork (TM) cells, contractile agonists such as bradykinin (BK) and endothelin-1 (ET-1) induce this type of Ca(2+) signaling. Given that trabecular outflow is modified by tissue contractility, the authors characterized SOCs and studied their participation in TM cell contractility. [Ca(2+)](i) was measured in cultured bovine TM cells loaded with Fura-2. Ca(2+) currents were recorded using the patch clamp technique. Cell contractility measurements were assessed by traction microscopy. BK and ET-1 activate a store-operated Ca(2+) entry that was greatly reduced in the absence of extracellular Ca(2+) or by preincubation with SOC blocker 2-APB or SKF96365. Store-operated Ca(2+) currents were also activated by intracellular dialysis with IP(3) + EGTA or after stimulation with thapsigargin. Electrophysiological characterization supports the presence of Ca(2+) release-activated Ca(2+) channels (CRACs) and nonselective cation channels, of which TRPC1 and TRPC4 channels may be candidate TRPs detected in TM cells. Extracellular Ca(2+) entry through SOCs is not required for TM cell contraction in response to BK or ET-1, but it modulates this process. Extracellular Ca(2+) entry in TM cells in response to agonist stimulation and store-depletion is mediated by the activation of SOCs, which do not contribute to cell contraction but which may activate regulatory mechanisms to prevent excessive contraction. CRAC and TRPC channels involved represent interesting modulators of TM function to improve aqueous humor outflow.

Profilin induces lamellipodia by growth factor independent mechanism

Profilin has been implicated in cell motility and in a variety of cellular processes, such as membrane extension, endocytosis, and formation of focal complexes. In vivo, profilin replenish the pool of ATP-actin monomers by increasing the rate of nucleotide exchange of ADP-actin for ATP-actin, promoting the incorporation of new actin monomers at the barbed end of actin filaments. For this report, we generated a membrane-permeable version of profilin I (PTD4-PfnI) for the alteration of intracellular profilin levels taking advantage of the protein transduction technique. We show that profilin I induces lamellipodia formation independently of growth factor presence in primary bovine trabecular meshwork (BTM) cells. The effects are time- and concentration-dependent and specific to the profilin I isoform. Profilin II, the neuronal isoform, failed to extend lamellipodia in the same degree as profilin I. H133S, a mutation in the polyproline binding domain, showed a reduced ability to induce lamellipodia. H199E, mutation in the actin binding domain failed to induce membrane spreading and inhibit fetal bovine serum (FBS) -induced lamellipodia extension. Incubation with a synthetic polyproline domain peptide (GP5)3, fused to a transduction domain, abolished lamellipodia induction by profilin or FBS. Time-lapse microscopy confirmed the effects of profilin on lamellipodia extension with a higher spreading velocity than FBS. PTD4-Pfn I was found in the inner lamellipodia domain, at the membrane leading edge where it colocalizes with endogenous profilin. While FBS-induced lamellipodia formation activates Rac1, PTD4-Pfn I stimulation did not induce Rac1 activation. We propose a role of profilin I favoring lamellipodia formation by a mechanism downstream of growth factor.

Forskolin Induces Myosin Light Chain Dephosphorylation in Bovine Trabecular Meshwork Cells

Purpose: Enhanced contractility of the actin cytoskeleton in trabecular meshwork (TM) cells is implicated in increased resistance to aqueous humor outflow. In this study, we have investigated effects of forskolin, which is known to elevate cAMP and also enhance aqueous humor outflow, on myosin light chain (MLC) phosphorylation, a biochemical marker of actin contractility. Methods: Experiments were performed using cultured bovine TM cells. Phosphorylated MLC (pMLC), expressed as the % of untreated cells, was assessed by urea-glycerol gel electrophoresis and Western blotting. RhoA activity was determined by affinity precipitation of RhoA-GTP to RhoA binding domain of an effector of RhoA. Intracellular cAMP levels were measured by ELISA. Results: Exposure to LPA (lysophosphatidic acid) led to increased MLC phosphorylation (LPA: pMLC = 133%) and activation of RhoA. These responses of LPA were suppressed by co-treatment with forskolin (LPA + forskolin: pMLC= 88%). Similarly, ET-1 and nocodazole-induced MLC phosphorylation (ET-1: pMLC = 145%; nocodazole: pMLC = 145%) as well as RhoA activation were suppressed by co-treatment with forskolin (ET-1 + forskolin: pMLC = 99%; nocodazole + forskolin: pMLC = 107%). Exposure to forskolin alone led to MLC dephosphorylation (pMLC = 68%). Forskolin alone led to a 4-fold increase in cAMP levels. This increase was not affected when co-treated with LPA or ET-1. Conclusions: Forskolin prevents MLC phosphorylation induced by LPA, ET-1, and nocodazole through inhibition of RhoA-Rho kinase axis. MLC dephosphorylation and consequent relaxation of actin cytoskeleton in TM cells presumably underlies the increased outflow facility reported in response to forskolin.

Effect of tTG AS-ODN on the expression of fibronectin in bovine trabecular meshwork cells

目的 观察组织型谷氨酰胺转移酶(tTG)AS-ODN(AS-ODN)对体外培养牛眼小梁细胞(BTMCs)纤维连接蛋白(FN)表达的影响.方法 设空白对照组、tTG AS-ODN1组和tTG AS-ODN2组.在脂质体介导下,将0.3μmol/L的tTG AS-ODN转染BTMCs,利用免疫组织化学SP染色法检测BTMCs表达FN情况,采用半定量RT-PCR和Western blot方法检测表达的变化.结果 免疫组织化学结果显示,AS-ODN1和AS-ODN2组灰度均值与空白对照组相比差异有统计学意义(P＜0.01),AS-ODN1和AS-ODN2组差异无统计学意义(P=0.91).RT-PCR结果显示,AS-ODN1和AS-ODN2组FN/β-actin条带中心密度比与空白对照组相比差异有统计学意义(P＜0.01),AS-ODN1和AS-ODN2组差异无统计学意义(P=0.73).Western blot结果显示,AS-ODN1和AS-ODN2组FN/β-actin灰度比值与空白对照组相比差异有统计学意义(P＜0.01),AS-ODN1和AS-ODN2组差异无统计学意义(P=0.92).结论 tTG可以促进BTMCs表达ECM.通过tTG AS-ODN抑制BTMCs表达FN等细胞外基质(ECM),有望从基因水平上防治原发性开角型青光眼(POAG)。

Synthesis and secretion of transferrin by a bovine trabecular meshwork cell line

The trabecular meshwork (TM) is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB) synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not) were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.

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.

Inhibitory effect of tissue transglutaminase (tTG) antisense oligodeoxynucleotides on tTG expression in cultured bovine trabecular meshwork cells

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides (tTG-ASDON) on tTG expression in cultured bovine trabecular meshwork cells (BTMCs) in vitro and explore a new treatment alternative for primary open angle glaucoma (POAG), the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON and tTG-ASDON2 were significantly decreased as compared with that of the controls (P < 0.05). On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.

Regulatory effect of dexamethasone on aquaporin-1 expression in cultured bovine trabecular meshwork cells

To evaluate the effect of dexamethasone on the expression of aquaporin-1 (AQP-1) in cultured bovine trabecular meshwork cells, bovine trabecular meshwork cells were cultured in vitro and reproduced to the third and the fourth generation, then treated with dexamethasone at the concentrations of 5, 25, 50, 250 microg/L respectively for 7 days. Immunohistochemical technique-supervision method was employed to measure, and image analysis system to analyze the expression of AQP-1 in normal cultured bovine trabecular meshwork cells and those treated with dexamethasone. In normal bovine trabecular meshwork cells, the grayscale of AQP-1 positive staining was 167.94 +/- 1.18, while it was 168.92 +/- 0.91, 176.72 +/- 1.80, 180.64 +/- 1.31, 185.64 +/- 1.58 in cells treated with 5, 25, 50, 250 microg/L concentrations of dexamethasone. When the concentration of dexamethasone was higher than 25 microg/L, the expression of AQP-1 was significantly inhibited (P < 0.05). The regulation of AQP-1 expression by dexamethasone in cultured bovine trabecular meshwork cells in vitro may be one of causes that retard the aqueous outflow in glucocorticoid- induced glaucoma.